BOLO Biology Newsletter Archive

Introduction to Eukaryotic Gene Regulation

2018-02-07T12:08:00.001-05:00

The Lac operon, a classic example of bacterial gene regulation, and set the stage for understanding Eukaryotic gene regulation. While Eukaryotes can possess the equivalent of operators, there are more complex levels of regulation to help determine the differentiation and physiology of cells. As we move to eukaryotic cells, we find that there are other ways that gene expression can be regulated. Below are a few examples. If you want to learn more about Eukaryotic Gene regulation, this page from the NCBI Bookshelf gives a good overview: Regulation of Transcription in Eukaryotes.

Transcriptional control: This is control of the promoter and operator, and is very similar to what was seen with the Lac operon.
Enhancers and Activators: Enhancers are sequences on the DNA that are found away from the initiation site (promoter). Activators are proteins that bind to enhancer sequences and help regulate the RNA Polymerase complex. This allows for variable expression of the gene. Eliminating one enhancer does not abolish transcription, but can reduce the efficiency of transcirption.
Epigenome: This occurs in both prokaryotes and eukaryotes. In eukaryotes, the epigenome is noted by chemical changes to DNA and histone molecules that result in changes to the chromatin strand. These changes can "lock down" genes, preventing even the recognition of the promoter.

IMPORTANT: the epigenome does not represent mutations. Instead, it is a reversible chemical alteration to chromatin structure.
This chromatin alteration can be passed vertically to offspring.
Changes to the epigenome occur due to chemical signals and environmental changes (they change the organisms adaptation range).

DNA Methylation (Epigenetic): Methylation of cytosine (the nitrogenous base of the nucleotide) prior to a promoter reduces transcription rates of the gene, or can inactivate the gene.
Post-Transcription regulation: If you don't add the 5' cap, then RNA can't leave the nucleus. If you don't add the 3' poly-A tail, RNA is digested. If you don't splice out the introns, you don't have a code. Prevention of post-transcriptional modification can play a role in gene expression.

Epigenome Videos

GOAL
At present, I want you to concentrate on gene regulation at the promoter. This includes the concepts discussed in regards to the Lac and Trp operons, as well as enhancers and activators. Having an idea of how chromatin remodeling works will also help in regards to eukaryotic gene regulation. In subsequent semesters, these concepts will be the foundation for further exploration of gene expression.

lac operon

2018-02-06T13:21:00.000-05:00

One of the most well-studied gene regulation systems is the Lac Operon found in Escherichia coli. To understand this system, it is important to first understand that bacteria do not experience Transcription and Translation in exactly the same way as eukaryotes (the mechanics are very similar, but there are some distinct differences).
The core difference between prokaryotes and eukaryotes is the nucleus. Since bacteria lack a nucleus, transcription takes place in the cytosol. There is no need for a tag to get out of the nucleus, so there is no need for capping. Also, there are no introns, so no need for splicing. Basically, there is no processing of RNA to make mRNA. There is no pre-mRNA. When a gene is transcribed, the transcript is mRNA.

https://cnx.org/resources/0d9811947495ba69dfd93f4c655e0384deb5b73e/Figure_16_01_01.jpg

Since there is no membrane separating transcription from translation, you can couple these to processes. As mRNA is made, it can be translated (polyribosome).

Bacteria have a single circular molecule of DNA (a genophore, not a true chromosome). They have to conserve their genetic space, so bacteria combine genes for a metabolic process in a single mRNA. IMPORTANT: bacteria can combine genes for a metabolic pathway into a sequential sequence with a single promoter. Thus, when you transcribe, you get all the genes for a given metabolic pathway.
The word OPERON describes this unique arrangement of prokaryotic genes: One promoter and one operator for a given series of metabolically linked genes.
The lac Operon holds three genes that give the cell the ability to take in and use the sugar LACTOSE. [Image Note: Bacteria have two promoter regions, -10 & -35, for a given gene or operon]

For E. coli, glucose is the preferred sugar. When glucose is present, there is no need to use lactose: these genes are not transcribed. When there is no glucose, E. coli has to use other sugars. IF lactose is present, the genes for lactose utilization will be made. Conversely, if there is no lactose, they genes remain locked down.

Glucose Present: No transcription
Glucose Absent: Minimal transcription
Glucose Absent, Lactose Present: Transcription of the lac operon.

NOTE: In this example, there are two ways to control the expression of a gene or operon:

You can block the operator of the gene. This prevents RNA polymerase from making RNA.
You can alter the promoter (or the interaction between transcription factors and DNA) to prevent binding of the Transcription Complex (RNA polymerase).

Author: Howard A. Shuman,Thomas J. Silhavy

https://www.nature.com/scitable/content/7005/10.1038_nrg1087-f1_full.gif

Negative Transcription Regulation (Repression) in the lac operon: There is a repressor for the lac operon. This is a protein that can bind to the operator of the lac operon (that region immediately downstream from the promoter). This creates a physical block that prevents RNA polymerase from transcribing.

The lac operon repressor (LACI) is a protein that is constitutively (always) expressed. This indicates that the lac operon is normally turned OFF. Notice that the gene for the regulatory protein is upstream from the lac operon. The lac repressor (LacI) binds to the major groove of the DNA at the operator. As you can see in the annotated image of the repressor, you have a DNA-binding region (active site), and a regulatory domain. The regulatory domain has the ability to bind to Lactose (the inducer).

You must have a way to unlock the operon, or to put it another way, to inactivate the repressor. An inducer is a ligand that can bind to a regulatory domain, changing the shape of the regulatory domain, and thus inactivating the DNA-binding domain. In the case of the the lac repressor, the ligand is allolactose (a derivative of lactose). When allolactose binds to the lac repressor, the repressor is inactivated, and the operon cleared. This is seen in the image below.

Therefore, the lac operon is partially regulated by the presence of lactose in the environment. If there is no lactose in the environment, then there is no need to transcribe the three genes needed to use lactose.

https://upload.wikimedia.org/wikipedia/commons/0/0c/Lac-operon.jpeg

Remember, we don't want to expend energy for things we don't need. Below is the size of the three gene products:

β-Galactosidase 1,024 Amino Acids
β-Galactoside Permease 418 Amino Acids
β-galactoside transacetylase 203 Amino Acids

Total Amino Acids, 1645. Using the assumption of 4 ATP per amino acid added to a protein, that makes 6580 ATPs needed just for protein synthesis. With this many amino acids, you are also looking at 4935 nucleotides (remember 3 nucleotides = codon = 1 amino acid), plus at least 3 stop codons. Each nucleotide added to a transcript takes the equivalent of 1 ATP, so you are looking at 4944 ATP minimum to make the transcript. Just to make one example of each protein (gene product), you are looking at 11,524 ATP. Do you just make one example of each protein? NO! Do you have the idea that this is energy consumptive? Would you make it if there was no lactose around?

Tomorrow we will look at the positive regulation of the lac operon. Remember, you still will not transcribe this operon if glucose is present. You only transcribe when glucose is absent. So how does the operon know when glucose is absent? That will be our discussion tomorrow.

Challenge

In your own words, describe how lactose (allolactose) is used to regulate the transcription of the lac operon. In your discussion, make sure that you explain the concept of an operon, and discuss the differences between eukaryote and prokaryote gene transcription.

The Lac operon has two levels of regulation. The first is through a repressor protein that binds to the operator of the operon. The repressor is constitutively expressed and binds to the Lac Operon operator. When lactose is present, it binds to the repressor, thus changing the shape of the repressor protein; the repressor detaches from the operator, and transcription can occur.

There is a second regulation system for the Lac Operon, and it has to deal with the promoter and the CRP binding site. In the case of the Lac operon, the promoter is "weak". RNA polymerase does not readily bind to the promoter. To assist in binding RNA polymerase, there is an activator site (CRP binding site) that can be used to "enhance" the promoter (enhance the binding of RNA polymerase to the promoter).

CRP stands for cAMP Regulator Protein (it is also known as the Catabolite Activator Protein). CRP has a binding site for cAMP, and the protein is activated (turned on) when cAMP binds. The CRP-cAMP complex can bind to the CRP binding site, and alter the Lac Operon promoter, enhancing the binding of RNA Polymerase to the promoter. But why this second regulatory system?

Remember that for this cell, glucose is the preferred carbohydrate and energy source (Escherichia coli is a chemoheterotrophic organism). The first regulatory system for the operon dealt with the presence/absence of Lactose (you only transcribe the operon when lactose is present). This second deals with the presence/absence of glucose. Glucose is the primary carbohydrate source, so as long as glucose is present, there is no need to transcribe pathways for secondary sugars.

In bacteria, the movement of glucose across the membrane (remember hexokinase?) inhibits the production of cAMP. If glucose transport slows dramatically or stops, Adenylate Cyclase begins making cAMP. So, while glucose available, there is little to no cAMP in the cell. When glucose is scarce, we see an increase in cellular levels of cAMP. For bacteria like E. coli, we can see cAMP as a starvation signal. The CRP-cAMP complex will bind to the bacterial DNA molecule and activate numerous pathways for alternative carbohydrate utilization. As with the Lac Operon, these other pathways will only transcribe when the correct sugar is in the environment.

Below is an image that demonstrates the two components of regulation.

https://commons.wikimedia.org/wiki/File:Lac_operon-2010-21-01.png

Below is a table that breaks down the regulation.

Challenge

Explain in your own words the evolutionary advantage of having a two-stage regulatory system, as seen in the Lac Operon. In your discussion, explain why it is important to have secondary messages like cAMP act as signals for large environmental changes, such as starvation states.

Introduction to Gene Regulation

2018-02-06T12:22:00.000-05:00

Study Skills

Reading textbooks and supplemental material is critical to becoming a successful student, as reading is an extremely effective way of learning if done actively. Active reading means you are critically engaged with the material covered in the textbook. You are forming questions, building summaries, and thinking about the topic. There are a number of active reading techniques out there, but they are not "one-size fits all". You need to experiment to see which technique works for you. As a note, give each of them a try; you won't know what works until you try it. Here is a video to help you consider different ways of reading the textbook.

Daily Challenge

Why do we need gene regulation? Today, reflect on the need and use of gene regulation. Why would an organism need to have some genes that it could turn on or off? Why would you need to control gene expression? Can the environment affect gene regulation? Can gene regulation affect evolution?

2018-02-01T11:42:00.000-05:00

Study Skills

It is always helpful to reflect on your study skills and see if there are ways you can improve your learning. Today, consider whether you maintain focus when you sit down to study.

Figure 1: chemical structure of an amino acid

Amino Acids and Polymerization

Amino Acids and Proteins Proteins will be a reoccurring topic throughout the semester, so for today let's first review amino acids and the basics of biochemical polymerization. They are one of the informational biopolymers. This means that they are composed of monomers (amino acids) that are linked together in specific sequences that are critical to their overall structure and function. [NOTE: It is important to understand the terms monomer and polymer, so make sure you have a good definition of these terms in your notebooks.] To understand proteins, we must first understand their monomeric unit, the Amino Acid.
Remember that all monomers will be chemically similar. In the case of the amino acid, the base molecule of Amino-Chiral Carbon-Carboxyl is the same. The difference in the amino acids comes with the side chain. These functional groups give each amino acid its unique identity and function. The twenty amino acids that are used in natural proteins can be found at the following link: Amino Acid Diagram. Notice that there are four general classes of amino acids with different chemical properties based on the functional group. NOTE: In the diagram to the right, R represents the radical group. This is the functional group or side chain. The distinctive characteristics of an amino acid are determined by the R group.

Figure 2: Polymerization of amino acids by a condensation reaction.

The base molecule is needed to link amino acids together into a polymer. A condensation (dehydration synthesis) reaction is used to form peptide bonds, the specific bond type that links amino acids together. [NOTE: biopolymers (save for lipids which are not polymers) have specific names for the bonds between monomers.] In the formation of a peptide linkage (bond), you will have a carboxyl and amino group linking together, with water being a product.

This linking of amino acids through peptide bonds will create the primary structure of a protein. All of the remaining levels of protein structure will result from interactions between functional groups on the amino acids. Local interactions induce folding into the secondary structures, which will result in other amino acids coming into close contact. This results in a tertiary structure. Finally, you will get a quaternary structure when multiple individuals folded peptide chains come together. Remember that Van der Waals forces (including hydrogen bonds), covalent bonds (disulfide bridges), and hydrophobic interactions will all induce folding. Because of this, environmental factors (such as heat or pH) can influence the folding pattern and shape of a protein.

Protein Self-Assemble (Folding)

Let's now further examine the importance of functional groups by discussing how they help form the working shape of a protein by causing a chain of amino acids to fold.

The primary structure of a protein is a chain of amino acids. Due to how proteins form, one end of the chain will end in an amino group (N-terminus), which the other end will have a carboxyl group (C-terminus). In Between these two terminal points will be a wide variety of amino acids.
The side chains of neighboring amino acids will begin to interact. They could be pulled toward each other, be repelled, or have nothing happen. Remember that the functional groups can twist around the chiral (central) carbon of the amino acid, so repulsion may just force the side chains to opposite sides of the chain (remember, you are dealing with 3-D structures here). The amino groups and carboxyl groups, even though they are part of the backbone, also retain polarity. Thus they can also be involved in the folding.

These interactions start the formation of the secondary level of protein structure. The two most common types of secondary structures are the alpha helix and the beta pleated sheet. These two types of secondary structures will help explain how the amino acid side chains start the folding process.

Figure 4: α-helix

Figure 4b: β-pleated sheet

In contrast, β-pleated sheet, shown on the right, has interactions between different regions of the primary structure. While only one amino acid chain is involved, in this case, the chain is not twisting. Instead, neighboring regions become attracted to each other. Also, the electrostatic interaction is between amino and carboxyl groups, not R groups. Multiple regions can be brought together to form these sheets as indicated in the diagram below. In this diagram, the red arrows represent a portion of the primary structure that has begun to form β-pleated sheets. Notice that six red arrows are arranged together. The purpose of this diagram is to show the placement of the β-pleated sheet. Consider a sheet. It is flat with two sides. Why do you think it would be important for a protein to fold in such a way as to create a relatively "flat" surface with two sides?

Figure 4a: β-pleated sheet

Notice that with the α-helix and β-pleated sheet we have altered the structure of the protein. It is no longer a linear chain but has greater dimensionality. We have either turned the protein into a rope/cord (α-helix) or into a "plane" with two faces(β-pleated sheet). Now areas that were once distant have been brought closer together. Now side chains can start interacting with each other.

Figure 5: Tertiary Structure

In the tertiary structure, different regions of the protein are brought into association. Electrostatic and hydrophobic interactions will force more conformational (structural) changes onto the protein. This will lead to a 3-dimensional structure. In the following diagram to the left, you can see an example of
a protein in primary and then tertiary structure.

Notice that the secondary structures are visible, but even these have been folded into each other.

Figure 6: Disulfide bridge

The forces that govern this are found in the R (functional) groups of the amino acids. Hydrophobic areas cluster together. Positive and Negative charges attract, while like charges repel. Polar partially charged side chains further interact, either with each other or with full charged. We also have a new interaction. The amino acid cystine contains a thiol (-SH). The thiols of two cysteines can react to form a disulfide bond. This is a covalent bond. Question: Which is stronger individually, a covalent bond or a hydrogen bond (electrostatic interaction)? The disulfide bond is utilized to stabilize the 3-dimensional structure of the protein.

Figure 7: Quaternary structure (Hemoglobin)

Many proteins are functional in the tertiary structure. Here you will see either globular or linear proteins (like collagen). There is a final level of structure. Some functional proteins are actually made up of multiple individual proteins. A great example of this is hemoglobin. The hemoglobin molecule, seen to the left, is composed of four individual proteins: 2 α-hemoglobin (red) and 2 β-hemoglobin (blue). The green structure is Heme, a prosthetic group that is used to hold oxygen and is attached by electrostatic interactions (van der Waals forces) to the proteins.

NOTE: Some proteins are functional in the tertiary structure, but others are only functional when you have multiple individual proteins forming the quaternary structure.
Denaturation: The protein is held together primarily through electrostatic interactions. What happens when a protein warms up? It starts to unfold. Why? The electrostatic interactions weaken as the kinetic movement of the atoms increases. Acids and bases, with their charged H⁺ and OH^- also disrupt these electrostatic interactions. Secondary and tertiary structures begin to change conformation. Most notably, they unfold. To denature a protein is to unfold it....but....
What if you only apply a mild heat, let's say your muscles warm up due to exercise. What happens to the proteins? What happens to hemoglobin when it passes through a warm temperature? Your muscles are also metabolizing, and as we will see, produce acids. What does this do? So, is denaturation all or nothing?

The tertiary and quaternary structures all have a specific electrochemical profile.
What happens if you add a charged particle/compound to a protein? What happens if I add a new positive charge? Answer: The protein will change shape (conformation).
What will this due to the function of the protein? It could actually activate the protein, but it could also deactivate the protein. This will be a discussion a little later in the semester, but I want you to start thinking about the implications.

Example: Hemoglobin
Adult hemoglobin is a quaternary protein, composed of two α-subunits (tertiary proteins) and two β-subunits (tertiary proteins). Each subunit contains a heme group which bears an Fe⁺². The heme is a prosthetic group of the individual proteins; without the heme, the subunit is non-functional. We will see further examples of prosthetic groups and other factors as we go through the semester. NOTE: We are looking at adult hemoglobin.

Figure 8: animation of hemoglobin t-r state transformation, made by en:User:BerserkerBen http://en.wikipedia.org/wiki/File:Hemoglobin_t-r_state_ani.gif


Figure 9: Normal vs. Sickle β-Hemoglobin http://helicase.pbworks.com/f/1225738609/hemoglobins.GIF

A well-known condition with hemoglobin is Sickle Cell Anemia. The condition is based upon a mutation of the gene that codes for the β-hemoglobin. The switch from an Adenine to Thymine causes a change in the primary sequence of the protein. In wild-type (non-mutant) β-hemoglobin, there is a glutamic acid (charged, negative) at position 6, but in Sickle Cell Anemia, the sixth position holds valine (non-polar). At low oxygen levels (such as in your tissues after exercise), the hemoglobin takes on an abnormal shape (

The quaternary protein has now changed shape by opening up. Sickle hemoglobin has the ability to bond to other sickle hemoglobins. This is due to hydrophobic interactions among valine, and the loss of a salt bridge (ionic interaction). The image below shows the result of this interaction between sickle hemoglobin: clumping.

Figure 10: Interaction between hemoglobin molecules
http://evolution.berkeley.edu/evosite/evo101/images/hemoglobin.gif

The clumping of sickle hemoglobin in low oxygen environments causes the distinctive change in shape seen in sickling red blood cells and is all due to changing one amino acid in the primary structure.

Below is an image comparing the shape of the normal erythrocyte (red blood cells) and the sickle variation.

Figure 11: Normal vs. Sickle Erythrocytes (Red Blood Cells)

Challenge:

Here are two challenges to help you think about protein structure.

Challenge 1

Hemoglobin is a protein that is constantly changing shape due to the presence or absence of oxygen. It also changes shape when in acidic environments or hot environments (think of your muscles after a work out). The effects of temperature and pH are described in the Oxygen Dissociation Curve. Temperature and pH disrupt electrostatic interactions, allowing for proteins to denature. Using the concept of denaturation, explain why more oxygen is released when the pH lowers and temperature increases. How would this be of benefit to the body?

Challenge 2

Antibodies help to defend the body against infections. They work by binding foreign chemical markers called antigens. The diversity of antigens is staggering, and cells that make them (B lymphocytes) have the ability to undergo somatic recombination (programed changes to DNA) to increase the variability of antibodies. Of specific importance is the variable domain:

Figure 12: Structure of an antibody
http://www.accessexcellence.org/RC/VL/GG/ecb/ecb_images/04_32_antibody.jpg

In the above diagram, look at the Variable Domain of the Light Chain (V_L). Notice 3 areas drawn in red, and referred to as the Loops that Bind Antigens. These are the areas where Somatic Recombination can change the nucleotides present in the gene. If I change the nucleotides (as with hemoglobin above), I can change the amino acids in the primary sequence. Explain how changing the primary sequence in these loop regions can change the binding affinity of an antibody to an antigen, and why this is beneficial to the body.

Translation

2018-01-31T14:42:00.001-05:00

The core concept of translation is the connecting of a codon to an amino acid, which is accomplished with the Transfer RNAs. What that leaves us with then is the actual mechanism of amino acid polymerization.

Initiation of Translation

Protein function is determined by the sequence of amino acids. This sequence allows the protein to fold into the correct configuration to produce activity. Any variation in the sequence can produce alterations to function, or even result in non-functionality. In order to generate the correct sequence, we must first establish the correct reading frame of codons. We must first find the start codon on mRNA.

The small subunit of the Ribosome (40s in eukaryotes) is built to find the Start Codon (AUG) and will align the full ribosome with the correct reading frame. A number of proteins will help the alignment and in the formation of the full (holoenzyme) Ribosome.
The diagram below shows the overall formation of the initiation complex, complete with a tRNA (the yellow structure with a pink circle attached). Again, the function of this replication complex is to find the start codon and set the reading frame for the Ribosome. Notice that the large ribosomal subunit (60s in eukaryotes) only attaches after AUG has been found. As before, it is not necessary at this academic level to memorize all of the factors involved. What is more critical is that it is a multifactor system designed to find the correct start point, and thus the same reading frame.

Key Feature: Notice that the first tRNA is already linked to the small subunit. Why? Its anticodon is complementary to the codon on mRNA. Specifically, it has the anticodon for the start codon (AUG). So we are using base complementarity to find AUG.

Elongation

The polymerization of amino acids occurs during elongation. This is where the P and A sites become important (NOTE: P and A sites are the active sites of the enzyme). P stands for Peptidyl, while A stands for Aminoacyl. These are chemical terms, which shows the orientation of the amino acid. The exit site, represented by E, is not an active site. Consider it a disposal point for spent tRNAs. [NOTE: you may also find references to a fourth site where the tRNA first comes into the complex. Don't worry about this optional site.]

The P and A sites reveal a single codon on mRNA and can hold a single complimentary tRNA. During elongation, when you have a filled P and A site, the amino acid from the P site will be linked to the amino acid in the A site. This is a process that you will have to visualize, so use the diagram below as reference:

https://karimedalla.files.wordpress.com/2012/11/translation_elongation.jpg

The amino end of the amino acid is free. The carboxyl end is attached to the tRNA. Starting at the top of the above diagram, the growing amino acid chain is attached to a tRNA in the P site. A new tRNA with an amino acid (charged tRNA) is brought into the A site. Using GTP, the Ribosome (large subunit) takes the growing peptide chain and links (carboxyl to amino) it to the individual amino acid in the A site. When this is done, the entire ribosome shifts downstream to the next codon (the new codon appears in the A site).

The spent tRNA that started in the P site is now moved to the E site, where it is removed from the ribosome. NOTE: It takes 2 GTP to create the peptide bond, then another GTP to move the ribosome. So a total of 3 GTP are used in one 'round' of Ribosomal action. REMEMBER THIS! In addition, it took a triphosphate to charge tRNA (so a total of 4 for each amino acid added to the polymer).
When both the P and A sites have charged tRNA, the growing chain from the P site is added to the single amino acid in the A site. The ribosome shifts and the process continues. This elongation process of adding amino acids (amino acid polymerization) will continue until a STOP codon is reached (UAA, UAG, and UGA).

Question: How much ATP will you need to expend to make a protein with 100 amino acids? How about a 150 amino acid protein? GOAL: Recognize and be able to articulate why protein synthesis is an energy consumptive process, and be able to discuss why it is critical for cells to regulate energy consumptive processes.

Termination

To create a functional protein, translation must end with the appropriate amino acid. If translation stops to soon, the protein will be too short and may not bend (configure) correctly. If it is too long, then it may not bend (configure) correctly. Termination is a critical process. Termination begins when a STOP CODON (UAA, UAG, and UGA) is reached. In eukaryotes, a releasing factor is used to separate the ribosomal subunits. KEY CONCEPT: The stop codon signals the end of the coded message.

http://www.proteinsynthesis.org/wp-content/uploads/2013/06/protein-synthesis-steps-termination.jpg

Once completed, proteins can be further modified as fits their function (such as adding sugars). This is known as post-translational modification. The image below shows the posttranslational modifications needed in the production of insulin. Production starts with a ribosome-bound on the Rough Endoplasmic Reticulum (RER). Processing will occur in the RER and in the Golgi body. This is only one example of posttranscriptional modification, and a majority of proteins require such modifications before they are functional. [NOTE: as a general rule, there is a less extensive posttranscriptional modification in prokaryotes, but they have numerous proteins that do require modification].

Daily Challenge

In your own words, describe the process of translation. Discuss initiation, elongation, and termination. Make sure that you discuss the P and A site, as well as the importance of the start and stop codon. Afterwards, give a BRIEF discussion on how this is an energy consumptive process that needs to be regulated.

Optional Challenge: Genomics and Proteomics

Read the following articles:
A brief guide to genomics - NIH fact sheet
Transcriptome - NIH fact sheet
Proteomics
The genome can be seen as the genetic potential of an individual (think Genotype), while the proteome shows what is actually produced at a given time, under a given condition (consider this the phenotype). Provide a discussion of the importance of genomic and proteomic studies in modern biological research, and make sure that you provide a description of both the genome and the proteome of an organism.

Optional Video

This video deals with advanced topics, so I give it to you in case you are curious.

Codons, Anticodons & Amino Acids

2018-01-30T12:51:00.002-05:00

Translation is the process of "reading" mRNA and using the code to construct a protein. But what is the code? The nucleotide language of mRNA can be divided into codons. Three sequential nucleotides that represent a genetic (nucleotide) word. So, how do you read this code or nucleotide language?

mRNA showing Codons

In the image to the right, you have sequential nucleotides divided up into codons. Notice that AUG is listed as Codon 1. This is important! AUG is the Universal Start Codon. Nearly every organism (and every gene) that has been studied uses the three ribonucleotide sequence AUG to indicate the "START" of protein synthesis (Start Point of Translation).

As we will see tomorrow, it takes more than a start codon to initiate transcription, but for now, just remember that this is the codon that indicates the START point of the instructions on how to make a protein.
The start codon established the Reading Frame for translation. From the start codon, every three sequential nucleotides will be viewed as a codon. This is critical! Mutations can affect reading frames. For example, if a nucleotide is inserted between codon 2 and 3 (G G), would you have the same reading frame down stream? What if you deleted the first nucleotide of codon 4? What is the effect of changing the reading frame? What would happen to the resulting protein?

Insertions and deletions can change reading frames, but point mutations can also occur. In this case, one nucleotide is change to a different nucleotide. What would happen if the final nucleotide of condon 3 were changed to a C? To an A? How about the second nucleotide in codon 4? Change the U to an A, what happens?

Each codon is a "genetic word," and refers to a specific amino acid (thus changes to these words can result in changes to final proteins). The tRNA is the agent of translation. On one end of the tRNA, you will find an anti-codon. Anti-codons are complimentary to codons. Example: Codon 1 reads AUG. The corresponding tRNA would have an anticodon reading UAC. (Question: Would these be antiparallel?). Codon 2 reads ACG, so the anticodon would read UGC. Oppisite the anticodon, you will find a binding site for a specific amino acid.

Aminoacyl tRNA Synthase - Addition of an Amino Acid to tRNA
http://canmedia.mcgrawhill.ca/istudy3/books/0070741751/images/figures/bro41751_1115L_lg.jpg

An amino acid can be attached to the free 3' end of the tRNA. There is a class of enzymes capable of attaching an amino acid to a tRNA: Aminoacyl tRNA Synthetase. Below is a very basic cartoon of how an amino acid is added to a tRNA.
Note that an ATP is needed to complete the binding. There is an Aminoacyl tRNA Synthetase for each tRNA-Amino Acid combination.
Below is a diagram showing the pairing of codon to anticodon. The diagram also contains a version of the Genetic Code table, showing the relationship between codon and amino acid.

Note that three codons are referred to as STOP codons: UAA, UAG, and UGA. These are used to terminate translation; they indicate the end of the gene's coding region. What would happen if you lost a Stop codon?

Challenge

How the change of one amino acid caused the configuration change in the protein. Amino Acids are coded due to a codon. In Sickle Cell Anemia, there is a mutation that changes one amino acid: Valine (Val) is found in place of Glutamic Acid (Glu). If we look at the sequences, we find that at the sixth codon, the wild type reads GAG, but the sickle type reads GUG. This is a single nucleotide polymorphism. At the end is a video to explain SNPs (pronunciation: Snips)

Here is the mRNA code for the wild type (non-mutant) and mutant β-hemoglobin molecule.

https://thestrangeandspectacularworldofbiochemistry.files.wordpress.com/2013/03/sickle.jpg

Today, consider the consequence of a SNP. What would happen if it occurred in a Start or Stop codon. What would happen if an AAG upstream (before) the start codon had a SNP that changed the second nucleotide from an A to a U? What would happen if CGC changed to CGG? How about CAU to GAU?

After considering these, and looking at the video, what are some of the consequences of a SNP? How could a SNP either stop translation or prolong it? Are all of the results harmful, or can they be neutral?

SNP Video

Group Projects: History of Biology Timelines

2015-10-12T10:50:00.000-04:00

Below are links to biology timelines created by students in Principles of Biology I (BIOL 2107)

Group 1

Group 2

Group 3

Group 4

Group 5

Group 6

Group 7

Group 8

Group 9

Group 10

Group 11

Group 12

Update: Summer 2015

2015-05-18T12:52:00.000-04:00

Over the next few months, I am going to be turning the content of this blog into an eBook for my students. Once I'm done with the process, I'll post it here.

As part of the process, I will be moving all newsletters that were posted in my LMS over to this blog.

I will also be making some office mixes to add interactive content.

Special Newsletter Gibbs Free Energy and Transition State Energy (Reposting)

2015-01-15T13:45:00.001-05:00

Special Newsletter
Gibbs Free Energy and Transition State Energy

This goal of this newsletter is to help students navigate the complexity of Gibbs Free Energy (ΔG) and help avoid confusion about Gibbs Free Energy and Transition State Energy (Energy of Activation).

Gibbs Free Energy (ΔG) is a thermodynamics concept, and describes the thermodynamic potential of a system. It measures the "work" that can be obtained from a system, for us a chemical reaction, if that system is held at constant temperature and pressure. The goal is to determine whether a given reaction is spontaneous in the direction written, i.e., whether the reaction will proceed in the given direction without the input of energy.

Mathematically, Gibbs Free Energy is described as:
ΔG=ΔH−TΔS

Where:

ΔG The change in Free Energy
ΔH The change in Enthalpy
ΔS The change in Entropy

In biochemistry, the change in Enthalpy (ΔH) is the same as the change in internal energy. Remember that ΔG is calculated based on a set of given conditions: we are at constant temperature and pressure, and all variables have been taken into account (such as the solvent of the system). If you change anything, you change the system, and thus the ΔG.

Gibbs free energy is a measure ONLY of the difference in free energy of the products and reactants, and does not tell us about the rate of the reaction. It only tells us whether the reaction is Exergonic or Endergonic.

ΔG < 0 Exergonic: The reaction is considered spontaneous in the direction written.
ΔG = 0 The system is in equilibrium
ΔG >0 Endergonic: To carry out this reaction as written, there needs to be an addition of free energy (NOTE: this means that the reverse reaction is spontaneous).

Transition State Energy is not Gibbs Free Energy.
Gibbs Free Energy does not describe the path of transformation or the mechanism of transformation. It does not describe the rate of transformation. It only describes whether the reaction is spontaneous, non-spontaneous or at equilibrium.

The breakdown of the dissacharide sucrose to glucose and fructose has a ΔG of -5.5 kcal/mol. This is a spontaneous (Exergonic) reaction in terms of ΔG. Yet you can store sucrose in your kitchen and it remains sucrose. There is no spontaneous degradation into monosaccharides. Why?

Sucrose is a stable molecule. In order to force the breakdown (catabolism) of sucrose, we need to destabilize the molecule. This destabilization is the transition state of the reaction, and in a closed system, requires the input of energy. This is termed the Activation Energy of the reaction (you will also hear this described as the Transition State Energy). If we were to look at our breakdown of sucrose, we would see the following:

Sucrose ⇄ Transition State → Glucose + Fructose

The energy of the transition state is noted as either Ea or ΔG‡. These two expression are from different formula for calculating activation energy. ΔG‡ is used in a formula that relates activation energy to Gibbs Free Energy (the Eyring equation). In either case, the expression describe transition state energy (aka, activation energy).

In biochemistry, enzymes are used to reduce the activation energy (just like catalysts are used in chemistry). Catalysts and Enzymes reduce the activation energy ΔG‡; they do not alter Gibbs Free Energy (ΔG). This is a critical concept!

Transition state deals with the rate of the reaction. By lowering the activation energy (transition state energy), you increase the rate of the reaction. In essence, you are making the reaction more likely to happen. But the enzyme does not change Gibbs Free Energy.

Original Image from ChemWiki,
http://chemwiki.ucdavis.edu/@api/deki/files/10017/fREE_eNERgY_cHART.jpg?size=bestfit&width=471&height=294&revision=1

Take home message:

Gibbs Free Energy (ΔG) describes whether a reaction is exergonic or endergonic. It does not describe rate, and neither enzymes or catalysts will alter ΔG.

Activation Energy (ΔG‡) does not alter ΔG; it does not determine whether a reaction is spontaneous or non-spontaneous. Activation energy does help determine the rate of the reaction.

Daily Newsletter September 24, 2014 Redox and Coupled Reactions (Reposting)

2015-01-15T13:43:00.003-05:00

Daily Newsletter

September 24, 2014

Redox and Coupled Reactions

Energy harvesting will be our topic next week, so I wanted to spend a moment and talk about energy in biological systems. Energy is a word that is often thrown around in various disciplines, and most of us carry misconceptions about the world from colloquial (common) use of the word. Scientists define energy as the capacity to do work. This definition helps to simplify a complex issue, but it starts to get confused when we apply it to the complex sets of reactions that we see in living systems.
For example, as we saw yesterday, the work in phosphorylation occurs in creating the covalent bond between a phosphate group and a substrate, not the action of the substrate. With ATP, the emphasis was shifted from seeing ATP as a battery that powered reactions. Instead, your focus was drawn to the Phosphate group, and the electrostatic effect it would have when added to a molecule or protein.

Next week we will discuss Energy Harvesting. Like with ATP, I want you to focus your attention on a specific form of energy, instead of holding a nebulous concept. Today the focus will be on reducing potential. Central metabolism describes the oxidation of glucose, so what are we harvesting? Reducing potential. So what is reducing potential?

A simple definition is reducing potential describes the capacity of a compound to donate electrons. Chemistry has a strict definition involving measurements with electrodes, but for our purpose, the concept of donating electrons is what is important.

Remember the characteristics of life. You must maintain homeostasis, and this means repair. You have to build nucleic acids, lipids, carbohydrates and proteins. These biosynthetic pathways often require you to reduce substrates. To stay alive, you need a constant supply of electrons for reduction; you need reducing potential. If you don't get these high energy electrons for reduction, you die. We will also find that this reducing potential is needed for us to make ATP.

Redox reactions are vital to our survival. Redox reactions are coupled Oxidation and Reduction reactions. One compound is oxidized as the next is reduced.

Remember, the molecules undergoing redox have to be close/touching. But in relative size, a cell is huge compared to a simple molecule. We may harvest electrons (oxidation) in one part of the cell, but use the harvested reducing potential in another part of the cell (reduction). Remember, you don't have free electrons; you can't throw electrons across the cytoplasm. So, how do we couple reactions that may be separated spatially? We use carriers!

Electron carriers, like nicotinamide adenine dinucleotide (NAD+), accept electrons at the site of oxidation, and then donate electrons at the site of reduction. NAD+ is readily oxidized and reduced during metabolic reactions, and there is only a negligible loss of energy from the electrons carried (can we ever have NO loss of energy? why or why not?).
NAD+ is also classified as a coenzyme, meaning it must work with an enzyme to accept or donate electrons. NAD+ can not randomly go to a molecule and oxidize or reduce it; its action is regulated by enzymes. NAD+ then must bind to an enzyme that catalyzes an Oxidation, and NADH must bind to an enzyme that catalyzes a Reduction.

Specifically, we couple the reactions. NAD+ has a place to bind into the enzyme, many times next to the substrate. The NAD+ can then capture the eletron pair that is released from the substrate. Additionally, one of the hydrogens will bind to the electron carrier. When we move to the next reaction, NADH will bind with an enzyme, again normally next to the substrate in question. The NADH can then donate the electrons (and hydrogens to the substrate. Enzymes thus help to couple these reactions. NAD+ will not just pick up an electron from any source, and NADH will not just donate electrons to any source. It must be mediated by enzymes.

Daily Challenge

This week you must complete all four forums to be eligible for the weekly summary and summative quiz.
Completion requires that you start a discussion, that your discussion is a minimum of 150 words on topics and at a collegiate level of writing, and that you reply to 3 of your fellow students.
Forum Closes: Septembe 25, 2014 at 11:55pm

Action of nicotinamide adenine dinucleotide (NAD)
In the citric acid cycle is the following reaction:

In this reaction, malate is oxidized. How do you know? You know because NAD is reduced to NADH. Below is a ribbon model of the protein malate dehydrogenase. Within the protein, you will see two molecules of NAD represented as balls. NAD binds to the enzymes active site first, and then malate binds. Within the active site are both + and - amino acids.

Your task today, using the enzyme malate dehydrogenase, explain how enzymes work and explain how reducing potential is harvested from organic compounds.

Daily Newsletter September 23, 2014 Adenosine Triphosphate (Reposting)

2015-01-15T13:42:00.000-05:00

Daily Newsletter

September 23, 2014 Adenosine Triphosphate

You have most likely heard ATP referred to as the "energy currency" of the cell. In fact, your textbook uses this analogy: "Just as it is more effective, efficient, and convenient for you to trade money for a lunch than to trade your actual labor, it is useful for cells to have a single currency for transferring energy between different reactions and cell processes."
This is a lovely fiction that does not serve molecular biologists. It is a convenient expression, but it conveys a very serious misconception.

Nucleotide triphosphates (ATP, GTP, CTP, TTP and UTP) have their foundation in the nucleotide structure, with the addition of extra phosphate groups. Adenosine Triphosphate is the most prevalent nucleotide triphosphate, and is found as a cofactor in a number of enzymatic reactions. The picture below show the general structure of ATP.

NOTE: the nucleotide triphosphates use RIBOSE as the sugar. If the nucleotide triphosphate utilized deoxyribose, we add the letter "d" in front of the abbreviation; so dATP represents a deoxyribo-nuclotide. The only time the cell ultilizes dATP, dGTP, dCTP and dTTP is during the process of replication (DNA synthesis).

You have three phosphate groups, each with a negative charge, covalently bonded to each other. The phosphate groups naturally want to repel each other, but they are held together by one of the strongest bond types (covalent bonds). What does this mean? Molecular tension! But it must be noted that ATP is chemically stable. It does not spontaneously loose phosphates (if it did, you would also release heat). It takes enzymatic action to remove the phosphate (i.e., we have to break the covalent bond). When a phosphate is removed from ATP, it is generally attached to another molecular structure (enzymes, sugars, etc...). The exception to this will be in building nucleic acids.

Chemist vs. Biologist

In yesterday's newsletter, a distinction was made between the perspective of chemists and biologists. If you look in most books that deal with biology, you will find the following euqation for ATP:
ATP + H2O → ADP + Pi ΔG˚ = −30.5 kJ/mol (−7.3 kcal/mol)
This is a look at ATP hydrolysis in isolation. Do you remember how ΔG is calculated? Look at the units. kJ (kilo-Joules) or kcal (kilocalories). We are basing this on an isolated reaction and measuring heat. When you look at the ΔG when there are metal ions present, you get a different number. You should also be familiar with ΔG, or Gibbs Free Energy, which is a measure of the amount of work that can be acheived by the energy release. A ΔG˚ = −30.5 kJ/mol is big, and implies that there is a great deal of energy released (the molecule can perform work).

Biologists though recognize that ATP is not in isolation. The intracellular fluid compartment (cytosol) contains ions, and more importantly metal ions, especially Mg2+ (causes major changes in hydrolysis ΔG). Inside of cells, the ΔG˚ of ATP hydrolysis is higher, approximately -50 kJ/mol (-12 kcal/mol). If we released this much energy every time ATP was hydrolyzed, the cell would boil! But, we use this energy for something else: binding the phosphate to another substrate (phosphorylation). When we phosphorylate a compound, we are building a covalent bond between the phosphate and the compound. This requires energy.1 About half the free energy is going to be used to make this covalent bond between the phosphate and the substrate. What happens with the rest? The second law of thermodynamics tells us that some of it is lost (this is an energy transfer after all...we broke one bond to build another), but the bond it self will hold some as well. Remember, chemical bonds are Potential Energy. When phosphate is removed, there will be another change in free energy (every reaction has a ΔG).

So what is the misconception with "energy currency"?
To answer this, we need to ask two other questions: why do we think of ATP as having High Energy Bonds and what is enery to a cell?

Why do we think of ATP in terms of High Energy Bonds? The answer is in the discussion above. The phosphoanhydride bonds (covalent bonds between the phosphate groups) have a high ΔG when they are hydrolyzed. This is why they have been called "high energy bonds". But in biology, the energy is used to immediately allow the phosphate group to bind to a new substrate (Phosphorylation). Put another way, the work that is done is in forming the covalent bond between the phosphate and the new substrate! The ATP is NOT a battery that energizes a curcuit.

ATP is always used in coupled reactions.

What is energy to a cell? Ask yourself, in building bonds in chemistry, what is the energy? If you want to change the energy state in a molecule, what do you do? Isn't it all about the electrons. Ultimately, the energy cells really use will be found with electrons, specifically through redox reactions. We will see that reducing potential is the energy the cells are harvesting and storing. This will be our discussion tomorrow.

Now we come to the big question: What does ATP do?
The concept of ATP as an "energy currency" comes from ATP turning on enzymes or assisting an enzyme during a "power steps" in a metabolic pathways. But ATP does not add energy; it just rearranges charge distribution around a molecule (electrochemistry). Remember, the phosphate group is negatively charged (-2).

When you add a phosphate group to a protein, you change the electrical signature around that portion of the protein (same will be true of other molecules as well). This includes the ability to form new hydrogen bonds, which can alter both secondary and tertiary structures. What will happen to the protein? It will change shape (conformational change). The work that ATP enables (and again this relates back to the free energy) is it's ability to cause conformational changes and induce molecular tension (instabilities).

Above is a general diagrapm showing a phosphorulation event. Notice how the phosphate group has interacted through van der Waals forces to change the tertiary structure of the protein.

The image below is a variant example of how ATP interacts with proteins. In this case, we see the the interaction of Myosin and Actin during muscle contraction. Focus your attention on the myosin molecule in blue. Notice that the "head" of the myosin changes conformation (shape) as ATP binds and is hydrolyzed. This is an important concept. When ATP binds, and hydrolysis occurs, a conformation change occurs. When ADP and Pi leave, the molecule resumes its "resting" conformation. In this case, it is the binding of the entire ATP molecule (with multiple negative charges) and subsequent hydrolysis which causes the conformational change. It still comes down to changing the electrical profile of the molecule (NOTE: there are more conformational changes occuring than are seen in this).
.

As we will see in upcoming weeks, this change of shape is critical to enzyme function. You have already encountered this once before, with the Sodium/Potassium ATPase.

Daily Challenge

Today, I want you to discuss the function of ATP. Do not describe it as an energy currency, instead describe how the addition of phosphates cause a change in the electrochemistry of a protein, and how that affects the conformation of the protein. Use Myosin in muscle cells as your example. We have not covered Myosin, but it is a very easy model for how ATP acts.

This is an easy to follow walk through of the interaction of ATP and Myosin. The site also contains further references.
Myosin ATPase activity: the 'powerstroke' cycle

Reference

1. Berg JM, Tymoczko JL, Stryer L. (2002). Biochemistry. W H Freeman, New York. http://www.ncbi.nlm.nih.gov/books/NBK22399/, accessed on September 25, 2012

Special Newsletter: Gibbs Free Energy and Transition State Energy

2014-09-19T19:07:00.000-04:00

Special Newsletter
Gibbs Free Energy and Transition State Energy

ΔG The change in Free Energy
ΔH The change in Enthalpy
ΔS The change in Entropy

ΔG < 0 Exergonic: The reaction is considered spontaneous in the direction written.
ΔG = 0 The system is in equilibrium
ΔG >0 Endergonic: To carry out this reaction as written, there needs to be an addition of free energy (NOTE: this means that the reverse reaction is spontaneous).

Sucrose ⇄ Transition State → Glucose + Fructose

The energy of the transition state is noted as either E_a or ΔG^‡. These two expression are from different formula for calculating activation energy. ΔG^‡ is used in a formula that relates activation energy to Gibbs Free Energy (the Eyring equation). In either case, the expression describe transition state energy (aka, activation energy).

In biochemistry, enzymes are used to reduce the activation energy (just like catalysts are used in chemistry). Catalysts and Enzymes reduce the activation energy ΔG^‡; they do not alter Gibbs Free Energy (ΔG). This is a critical concept!

Transition state deals with the rate of the reaction. By lowering the activation energy (transition state energy), you increase the rate of the reaction. In essence, you are making the reaction more likely to happen. But the enzyme does not change Gibbs Free Energy.

Original Image from ChemWiki,
http://chemwiki.ucdavis.edu/@api/deki/files/10017/fREE_eNERgY_cHART.jpg?size=bestfit&width=471&height=294&revision=1

Take home message:

Gibbs Free Energy (ΔG) describes whether a reaction is exergonic or endergonic. It does not describe rate, and neither enzymes or catalysts will alter ΔG.

Activation Energy (ΔG^‡) does not alter ΔG; it does not determine whether a reaction is spontaneous or non-spontaneous. Activation energy does help determine the rate of the reaction.

Daily Newsletter: September 10, 2014 - Codons, Anticodons & Amino Acids

2014-09-09T14:14:00.001-04:00

Daily Newsletter

September 10, 2014

Codons, Anticodons & Amino Acids

Translation is the process of "reading" mRNA, and using the code to construct a protein. But what is the code? The nucleotide language of mRNA can be divided into codons. Three sequential nucleotides that represent a genetic (nucleotide) word. So, how do you read this code or nucleotide language?
In the image to the right, you hav
e have sequential nucleotides divided up into codons. Notice that AUG is listed as Codon 1. This is important! AUG is the Universal Start Codon. Nearly every organism (and every gene) that has been studied uses the three ribonucleotide sequence AUG to indicate the "START" of protein synthesis (Start Point of Translation).

As we will see tomorrow, it takes more than a start codon to initiate transcription, but for now just remember that this is the codon that indicates the START point of the instructions on how to make a protein.
The start codon established the Reading Frame for translation. From the start codon, every three sequential nucleotides will be viewed as a codon. This is critical! Mutations can affect reading frames. For example, if a nucleotide is inserted between codon 2 and 3 (G G), would you have the same reading frame down stream? What if you deleted the first nucleotide of codon 4? What is the effect of changing the reading frame? What would happen to the resulting protein?

Insertions and deletions can change reading frames, but point mutations can also occur. In this case, one nucleotide is change to a different nucleotide. What would happen if the final nucleotide of condon 3 were changed to a C? To an A? How about the second nucleotide in codon 4? Change the U to an A, what happens?

Each codon is a "genetic word," and refers to a specific amino acid (thus changes to these words can result in changes to final proteins). The tRNA is the agent of translation. On one end of the tRNA, you will find an anti-codon. Anti-codons are complimentary to codons. Example: Codon 1 reads AUG. The corresponding tRNA would have an anticodon reading UAC. (Question: Would these be antiparallel?). Codon 2 reads ACG, so the anticodon would read UGC. Oppisite the anticodon, you will find a binding site for a specific amino acid.

An amino acid can be attached to the free 3' end of the tRNA. There is a class of enzymes capable of attaching an amino acid to a tRNA: Aminoacyl tRNA Synthetase. Below is a very basic cartoon of how an amino acid is added to a tRNA.
Note that an ATP is needed to complete the binding. There is an Aminoacyl tRNA Synthetase for each tRNA-Amino Acid combination.
Below is a diagram showing the pairing of codon to anticodon. The diagram also contains a version of the Genetic Code table, showing the relationship between codon and amino acid.

Daily Challenge

In an earlier newsletter, we discussed Hemoglobin, and how the change of one amino acid caused the configuration change in the protein. Amino Acids are coded due to a codon. If you recall, Valine (Val) is found in place of Glutamic Acid (Glu). If we look at the sequences, we find that at the sixth codon, the wild type reads GAG, but the sickle type reads GUG. This is a single nucleotide polymorphism. Here is a video to explain SNPs (pronunciation: Snips)

Today, consider the consequence of a SNP. What would happen if it occurred in a Start or Stop codon. What would happen if an AAG upstream (before) the start codon had a SNP that changed the second nucleotide from an A to a U? What would happen if CGC changed to CGG? How about CAU to GAU?
After considering these, and looking at the above video, what are some of the consequences of a SNP? How could a SNP either stop translation or prolong it? Are all of the results harmful, or can they be neutral?

Link to Forum

Daily Newsletter: September 8, 2014 - Ground Rules for Gene Expression

2014-09-08T00:30:00.000-04:00

Daily Newsletter

September 8, 2014

Ground Rules for Gene Expression

(AKA Central Dogma)

Central Dogma, in broadest sense, encompasses the genetic mechanisms of Replication, Translation and Translation. In the strictest sense, Central Dogma describes gene expression: Information encoded in the nucleotides of DNA being use to construct proteins. The two core genetic processes involved in gene expression are Transcription (synthesis of RNA) and Translation (synthesis of proteins).

Central Dogma of Biology

Before digging into each process, let's talk a little about what is at stake here. DNA holds our genetic history. It holds codes on how to build an organism, but what does that really mean?

The basic unit of life is the cell, and cells are formed from phospholipids can naturally form bilayers. Furthermore, phospholipids can even natrually form spherical structures that create two fluid compartments, outside vs. inside. The phospholipids that make up the cellular membrane form the most basic feature of the cell: a dividing point, separating the inside from the outside (more on this in the weeks to come).

Membranes though are passive. As a selectively permeable barrier, only certain materials can cross. Proteins add functionality to the membrane. By embedding proteins, you can chance the permeability of the membrane. This is how cells balance what is on the inside, and what is on the outside. Membrane proteins can also have enzymatic or signal functions. Proteins add functionality to the membrane.

A common expression is that DNA holds the code to make an organism. The meaning of this phrase lies in the concept that by making proteins, we make phospholipid membranes and cells functional. From DNA, cells can build proteins for metabolic pathways, to produce various chemical compounds, anchor with other cells, and in multicellular complex life, we even have the development of special cellular roles that work together to form a composit whole.

The concept of how we go from DNA to RNA and then Proteins is one of the most critical concepts in biology! Today we are going to focus on some of the basics, the Ground Rules, of genetics.

All genetic processes work due to base complementarity. If you know the base complementarity rules, then the foundations of genetics will make sense. At times, this may seem repetitious, but I really want you to get these terms and concepts.
Genes are sometimes referred to as the unit of heredity, and with good reason. A gene is a segment of DNA that holds the code to make a protein (NOTE: or functional RNA, such as trasfer RNA). In modern biology, we refer to gene products, which are just the expressed macromolecules coded by a gene.
Remember, a gene product can be either a proteins or functional RNA (e.g., tRNA). Functional RNA does not code for proteins, instead, these RNA strands have some function in cellular metabolism, most notably in the genetic process of Translation. Examples include tranfer RNA (tRNA), ribosomal RNA (rRNA), and small nuclear RNA (snRNA).

All genes have non-coding portions that are critical for the correct transcription (synthesis of RNA). These non-coding areas are critical for regulation and aligning the transcription enzymes (e.g., RNA polymerase). Below is a graphic that shows structure of a gene. The promoter of a gene is a sequence of DNA upstream of the actual code (coding region) that indicates the "Start" point for transcription. This is how your cell knows where to begin transcription. The loss of the promoter means that the gene will no longer be expressed.

In Eukaryotic cells, a common promoter is a DNA sequence that reads TATAAA, and is better known as the TATA-Box. In bacteria the promoter is known as the Pribnow Box (Pribnow-Schaller box). In both cases, the promoter is found in the Major Groove of the DNA molecule. As can be seen in the image to the right, the major groove is wide enough to "see" the base pairs. The base pairs have an electrochemical profile, and thus can respond to other chemicals (via van Der Waals forces). Thus, the major groove is a place where proteins (and other compounds) can bind to specific sequences of DNA! The promoter sequences are found in the major groove. The image to the right shows a bacterial promoter event. One of the factors needed to start transcription (by recognizing the promoter) has bound into the major groove. This recognition event is needed to identify the start point of a gene. The Transcription Initation Complex will then begin to form at this site, and begin the transcription of the gene.

Many genes are regulated, meaning they can be turned on and off. Beyond a promoter, a regulated gene will typically have a non-coding region known as the Operator. The operator is located down stream of the promoter (meaning it will be between the promoter and the coding region). Regulatory proteins can bind to the operator, preventing transcription. Remember, cells are masters at energy conservation. They will not begin producing proteins that are unnecessary. Gene regulation is a common activity of Signal & Receptor systems. The image below is a good visual of the promoter & operator systems. House keeping genes are those that are needed for the general function of the cell, and can include genes for glycolysis, citric acid cycle, and ribosomes. These genes are always ON, and are referred to as constituative genes.

Messanger RNA (mRNA) is a molecule of RNA that cares the gene code for the construction of a protein. mRNA is sent to the Ribosome in order to produce a protein. The code for constructing a protein is in Nucleotide Language, meaning the code is a code of nucleotides. Specifically, the code in mRNA is in the ribonucleotide language (A, U, G, C). In order to make a protein, it is necessary to Translate the ribonucleotide language into the language of proteins, i.e., amino acid sequcences.

In order to translate, you need an agent of translation. This agent of translation must be a molecule that contains both ribonucleotides and amino acids (think of it as the nucleotide-amino acid dictionary). A specific ribonucleotide sequence must directly correspond to an amino acid, just as in translating human languages requires word for word relationships. This concept of a direct nucleotide to amino acid relationship is the basis of the Genetic Code.

The agent of translation is Transfer RNA (tRNA). In tRNA, there is a direct physical correspondence between a 3 nucleotide sequence (anti-codon) and an amino acid. To the right are common ways of illustrating tRNA, with the 3^rd image being the most common way of drawing the molecule. In the image, each molecule has a region known as the anticodon; this region will interact with mRNA. At the 3' end of the molecule, a specific amino acid will be bound.

On the mRNA, the code is broken down into codons (think of these as genetic words). Codons consist of 3 adjacent nucleotides. Codons are complimentary to anticodons found on tRNA. Each tRNA has a specific anticodon-amino acid relationship, so each codon then specifies an amino acid. The genetic code is NOT ambiguous. There is a direct correspondence between codon and amino acid; the tRNAs make sure of this.

The ribonucleic language is divided into 64 3-nucleotide words known as codons. Condons specify though tRNA an amino acid. The Genetic Code is thus the translation scheme between codons and amino acids. [NOTE: another way to describe the genetic code is in terms of a computer algorithm]. Below is a rather unique way of viewing the genetic code. It is an excellent way of visualizing the number of redundancies in the code.

The genetic code is redundant, which means that there are multiple codons (3 nucleotides) that specify the same amino acid. For example, around the 12 o'clock position of the above chart, you see the amino acid glycine. The codons GGU, GGC, GGA and GGG all specify Glycine. Phenylalanine is specified by UUU and UUC. There are only a few amino acids, such as methionine, that are specified by a single codon (in the case of methionine it is AUG).

The presence of redundancies means that some alterations in the gene sequence are silenced (silent mutation). For example, changing GGU to GGA does not change the specified amino acid (Glycine). This is a silent mutation. Changing UUC to UUA may cause a problem (point mutation), but both Leucine and Phenylalanine are hydrophobic, so the variation may be minor. Chaing CAC to CAG though has more impact as you are changing the positive histidine to a polar glutamine (you loose the full positive charge of histidine). Remember, chaning amino acids can easily change the way a protein folds. REMEMBER: The genetic code has redundancies, and this will limit some problems with mutation.

Below is a more classic way to represent the genetic code, in the form of a table. The way the table is arranged, you can easily see the various redundancies in the system. In both representations, notice that there are three codons that specify STOP. These stop codons, UAA, UAG and UGA are essential for the termination of protein synthesis. In the image below, you will notice AUG has been tagged as the initiation (start) codon. All protein synthesis begins with the code AUG. We will talk more about this later in the the week.

Daily Challenge

Today's newsletter helps to set the stage for our discussion of the central dogma of biology (gene expression). In reading you find that DNA hold the codes to make various types of RNA and Proteins. Most of the time, what concerns us is the production of proteins, as they will add functionality to our cells.
At the heart of the Central Dogma is the genetic code. This code shows how you move from the language of nucleic acids to the language of proteins (aka, amino acids). This code is Universal and Non-Ambiguous, but what does that mean? Your goal today is to read, in your text and in the optional reading, and reflect on the concept of gene expression and the genetic code. Why is it so important? How do we use it? How does this influence concepts from understanding hormonal changes at puberty, evolution and genetic engineering?

Link to Forum

Daily Newsletter: September 5, 2014 - DNA Replication

2014-09-05T11:38:00.001-04:00

Daily Newsletter

September 5, 2014 - DNA Replication

Textbooks have a tendency to make replication one of the most complext topics covered. With a tendency to throw all the current research and understanding at students, they rarely take a step back and try to explain it. This newsletters has two goals: 1) to help biology students understand DNA replication, and 2) to show you want is expected from a Biology Freshman/Sophmore.

Central Dogma of Biology

Before we get into replication, let's take a step back and look at the three core genetic processes, a.k.a., the Central Dogma. The central dogma describes the flow of genetic information in a cell. The core idea is INFORMATION. You may recall some of our early discussion on DNA, and about base complimentarity and the directionality of the molecule. This will become important rather quickly, but for just this moment, I want you to concentrate on the fact that DNA carries information. Information on how to build RNA and Proteins, both of which will produce the phenotype (expression) of the cell. For this reason, DNA, RNA and Proteins are considered Informational Macromolecules. This means that the sequence of monomers contains infomation, e.g., instructions on how to build RNA and Proteins. Since it is critical, the sequence of nucleotides carries information.
As you can see from the diagram of central metabolism shown above, there are three processes: Replication, Transcription and Translation. Think about those words. They are words used in reference to languages and documents. When you replicate a docuement, you want to ensure that you are getting a faithful (or even exact) copy of the original.

When you transcribe, you are moving from one medium, e.g., spoken word, to another medium, e.g., text. If you watch news shows, they will tell you that transcripts of the show are available. Court reports make transcripts of the trial. You are taking the language from one medium (in our case DNA) to another medium (RNA). The language is still the same (i.e., nucleic acid lanugae), just in a different form. Does the transcript have to be 100% correct? You want it to be, but it is not as exacting as a replication.
Translation is where you change languages. Unless you're fluent in another language, you will need somoene to help translate, or at least a good translation dictionary. Now you are moving the context from one language (nucleic acid) to another language (amino acids).

All of these processes rely on one key feature of nucleic acids: BASE COMPLIMENTARITY. In DNA: A complements T, and G complements C. In RNA: U complements A, and G still complements C.

Replication

In replication our goal is to take one molecule of DNA and make two daughter molecules of DNA that are identical to the first. Even the best replication processes can produce errors, but our goal is to be error free. This takes precision! As DNA is long, we also need this to be a fast process.

The enzyme that is ultimately responsible for replication is DNA Polymerase. [NOTE: there are multiple types of DNA Polymerase, but for now you just need to understand the core concept common to the DNA Polymerase family.] DNA polymerase is only one component of the Replication Complex, which is a complex association of proteins needed to successfully complete the replication event. Your goal at this time is to concentrate on DNA polymerase; we will talk about some of the other components later.
DNA is a double stranded molecule, in which the strands are anti-parallel. This means that one strand starts at the 5' end → 3' end, while the the other strand is revered. This can be seen in the image to the right. A common way of saying this is that we read DNA in the 3' → 5' direction. Why is this important? DNA Polymerase can only read DNA in the 3' → 5' direction, and can only build a new strand in the 5' → 3' direction. Before we go on, let's look another time at how deoxyribonucleotides are polymerized.

To the left you will see a generic image showing DNA Polymerase adding nucleotides to a growing DNA strand. Look carefully: A deoxyribonucleotide triphosphate (dTTP) is being added. The 5' phosphate of the new nucleotide is what will be used to form the phosphodiester bond. DNA Polymerase requires a free 3' end on which to grow the new DNA strand. To the right is another image that will help you with this concept. At this point, the critical thing to remember is that DNA Polymerase will need a free 3' end on which to add a new nucleotide.
This requirement to build in only one direction (5' → 3') creates a problem for the DNA process: the two strands read in opposite directions, and each must be replicated. It was noticed that one strand appears to replicate continuously, while the opposite strand appears to replicate discontinuously.

The original strand that reads 5' → 3' can be used by DNA polymerase to continuously produce the new 5' → 3' strand (the antiparallel complement to the original strand). We refer to the continuous synthesis strand as the Leading Strand. The other original strand, which read 3' → 5' cannot be copied continuously. A section will have to be exposed, replicated, and then another section exposed. This strand is constructed discontinuously, and is reffered to as the Lagging Strand.The image above shows the leading and lagging strand. Notice that the leading strand is replicating toward the Replication Fork(where the original strands seperate). As more DNA unwinds and opens, DNA synthesis continues down the leading strand.
The lagging strand though has to build in a start-stop action, producing Okazaki fragments. These fragments have to be sealed (phosphodiester bonds) together before DNA can rewind into the α-helix.

In the image above, you will notice a number of enzymes on the lagging strand. These enzymes are required for the initiation of DNA polymerazation, and then sealing the fragments. Looking at the image, you will see an enzyme called Primase, and a structure in red known as a Primer. Another restriction on DNA polymerase is that it must have a free 3' end from which to start building. DNA Polymerase is prevented from building a DNA strand from nothing. Something (a primer) must be in place upon which DNA polymerase can build. The Primer is constructed from RNA, and is a temporary scaffold upon which DNA polymerase can start working.

Eventually the primer will need to be moved. This is where you need to learn a little more about DNA polymerase. There are multiple DNA polymerases in eukaryotic systems. The general work horse of replication is DNA Polymerase III (DNA pol III), which is used to make long strands of DNA. DNA Polymerase I (DNA pol I) is used to replace primers (it is also used in repair functions). Even after the RNA primer is replaced with DNA, there is still a gap between fragments. Ligase is the enzyme used to create a phosphodiester bond between fragments, thus sealing the new sugar-phosphate backbone of the synthesized strand.

The result of DNA replication is that one molecule one DNA was used to create two new molecules of DNA. The two strands of the original DNA molecule became the template from which to build new complimentary strands of DNA. This is referred to as semi-conservative replication, as each new molecule has one strand from the original molecule, and one freshly synthesized complimentary strand.

Daily Challenge

In your own words describe the purpose and process of replication.
Link to Forum

Daily Newsletter: September 4, 2014 - Nucleotides

2014-09-04T14:01:00.003-04:00

Daily Newsletter

September 4, 2014 Nucleotides

Nucleic Acids

Nucleotide Structure: The following image from wikipedia's image gallery shows the basic structure of the nucleotide and the five nitrogenous bases.

The central component of all nucleotides will be a pentose sugar (5-carbon sugar). We will either see ribose or 2'deoxyribose as the sugar (the second carbon has one less oxygen than ribose). Off of the 5' carbon of the sugar, you will find a phosphate group attached, while on the 1' carbon, you will find a nitrogenous base. [NOTE: remember the numbering of carbon atoms in carbohydrates from yesterday? Do you see why the numbering is important?]
There are five nitrogenous bases, divided into two categories: Purines and Pyrimidines. Notice that the purines are a composite of two ring structures, while the pyrimidines are a single ring structure. When you take organic chemistry and biochemistry, the importance and complexity of these ring structures will be further discussed. At present, just become aware of their respective shapes and sizes (and inclusion of nitrogen).

As with amino acids, the nucleotide contains a functional group: the nitrogenous base. Just like the side chain in an amino acid, the nitrogenous base will play an important part in the function of this biomolecule. The Sugar-Phosphate then becomes the backbone of the molecule (line the Amino-Chiral Carbon-Carboxyl of an amino acid). We will in later weeks that the sugar-phosphates of nucleotides will create the strands of DNA and RNA. The nitrogenous bases then playing an information role.

Base Complementarity:

The nucleic acids are referred to as informational biomolecules (biopolymers). This is because the sequence of nucleotides carries information on how to build RNA and Proteins. One of the central foundations of genetics (i.e., how it all works), is base complementarity. Here we are looking at the interactions between purines and pyrimidines:

A links with T through 2 hydrogen bonds.

G links with C through 3 hydrogen bonds.

A to T G to C

U has the binding properties of T, but is only found in RNA.
T is never found in RNA, only DNA.
NOTE: base complementarity is a critical concept to remember. All genetic processes rely on base complementarity!

Directionality

When we get to genetics, we will be talking about the directionality of the nucleic acids. For example, we will talk about DNA being built from the 5' to 3'. This is in reference to the carbon atoms in the ribose or deoxyribose. The 5' holds a phosphate, while the 3' holds an open -OH (hydroxyl) group. This concept of directionality is critical, and you are warned to learn how it works, and what the terms represent.
As with all biopolymers, monomers are added together through dehydration synthesis, and separation is through hydrolysis. When synthesis occurs, the 5' phosphate links to the 3' -OH, forming a phosphodiester bond.

Daily Challenge

The challenge today is to understand the history of the discovery of DNA. Look up the following researchers and read about their discovery, how it was done, and the importance the the discovery. In addition, watch the TED Talk from James Watson "How we discovered DNA." Write up a discussion about how these researchers contributed to our understanding of DNA.
Link to Forum

Daily Newsletter: September 6, 2013 - Carbohydrates and Lipids

2014-09-03T11:55:00.003-04:00

Daily Newsletter

September 6, 2013 Carbohydrates and Lipids

Carbohydrates

While carbohydrates are mainly used as chemical energy storage, carbohydrates are also used as modifiers of proteins and in forming cellular receptors and anchors. One of your goals is to gain a good understanding of the structure of carbohydrates, and a little about their naming.

A topic that will come up throughout the semester is how carbons are numbered in carbohydrates. This is important as we will find carbohydrates being components of monomers and when we move through the carbohydrate catabolism. The following image from Rensselaer Polytechnic Institute shows the linear form of glucose, and the two possible cyclic (pyranose ring) isomers.

The formation is based on aldehyde chemistry, so we will leave some of this discussion to organic chemistry and biochemistry. For our purpose this semester, what is important is that we number carbons from the aldehyde. Notice in the above diagram that carbon 1 is to the left of the oxygen, we go around to carbon 5, and then carbon 6 is outside of the ring. If you see the expression 3', it is referring to the third carbon. 5' the fifth carbon. 6' the sixth carbon, and so forth.

Notice also, that when the ring was formed, there were differences in the groups coming off of carbon 1. These differences are important, and can influence how the sugar is metabolized. We say that these different forms are isomers (if you don't know what an isomer is, look it up and add the definition to your notebook).

One critical difference comes when linking two monosaccharides together to form disaccharides and polysaccharides. For instance (again from rpi.edu), here is maltose:

This is an α 1-4 glycosidic linkage. We have an α Maltose (look at carbon 1) bound from carbon 1 to carbon 4. Since the maltose on the left hand side is α at the 1 carbon, we form an α linkage. In comparison, look at cellobiose:

Cellobiose has a β 1-4 glycosidic linkage. The designation of β comes from the sugar unit that donates carbon 1 to the bond.

So, what is the big deal? Maltose is digestible by humans, cellobiose is not. Just this slight isomeric difference changes the metabolism.
All carbohydrate monomers are connected through glycosidic linkages, whether it is a disaccharide, oligosaccharide or a polysaccharide. Make sure that you learn the different types of carbohydrates.

Lipids

Lipids are an odd group of biomolecules. Proteins, Carbohydrates and Nucleic Acids are all formed through polymerization reactions; they have monomeric units that join to make polymers. Lipids do not polymerize, and they have no monomers. Instead, Lipid is a word that defines a class of hydrophobic organic compounds found in living systems. There are a number of important groups of Lipids, such as the triglycerides, phospholipids and cholesterols. Today, we are going to concentrate on the triglycerides and the phospholipids.
Both triglycerides and phospholipids possess a glycerol molecule and fatty acids. Glycerol is a 3 carbon compound that we will see from time to time. It acts as the backbone or schaffold of the triglycerides and phospholipids. As you can see, on each carbon atom, there is a hydroxyl group (-OH). This hydroxyl group is where other molecules can bond. Another thing to note is that there is free rotation around the carbon atoms. When you take organic chemistry, you will learn more about free rotation, why it is important, and how it can affect a molecule. For now, just note that there can be free rotation.
Fatty acids are long hydrocarbon chains with a carboxyl group (-COOH) at one end. To the left is a diagram of palmitic acid, a typical saturated fatty acid. In this diagram, each angle on the line represents a carbon atom, and off of these carbon atoms are hydrogens. This is one of many ways that organic chemicals are depicted. Below the first depiction is a molecular model. Carbon atoms are in black, and the white balls are hydrogens. What you will begin to recognize in these diagrams is that there are only single bonds between the carbon atoms. This means that the maximum number of hydrogen atoms are attached to the fatty acid. In other words, they are saturated with hydrogen.
In contrast, an unsaturated fatty acid does not have the maximum number of hydrogen atoms. This occurs when double bonds (two electrons from each carbon are shared) occur in the carbon chain. To the right is a diagram of oleic acid. Note that there is a double bond in the carbon chain. Notice that the chain is bent, or kinked. This creates a very different structure for lipids that carry unsaturated fatty acids.
As a general rule, saturated fatty acids are solid at room temperature, and unsaturated fatty acids are liquid in room temperature. But one thing is the same in both: the carbon chains are HYDROPHOBIC!
In a triglyceride, the carboxyl end of the fatty acid will react with the hydroxyl end of the glycerol. As you can see in the diagram, the two molecules are joined together through an oxygen molecule. As with other biosynthetic reactions, this is a dehydration synthesis (water is released). The resulting bond, as noted in the diagram, is an ester bond. You will learn more about this bond in organic chemistry, so for now I just want you to remember the general look of it. Why is this so critical? Because not all living organisms make triglycerides and phospholipids this way. Member of domain Archaea use an ether bond.
So, what is the difference between a triglyceride and a phospholipid? Recall the look of the glycerol, and note that there are three locations where an ester bond can be formed. In a triglyceride, a fatty acid will be bound to each of the carbon atoms by ester bonds. Tri- means three, so we have three fatty acids attached to the glycerol. The image to the right is an example of a triglyceride, and please note, you can have more than one type of fatty acid in a triglyceride.
The phospholipid in contrast only has two fatty acids. The third binding location will be used for a "phosphate head". This head contains a phosphate group and usually a diglyceride and some small charged organic structure. Phosphytidyl choline is a commonly studied phospholipid that uses choline as the charged organic structure. NOTE: both the phosphate group and the organic structure carry a charge. This phosphate head is charged, thus it is hydrophillic (water loving). The phospholipid contains non-polar, hydrophobic fatty acids (usually referred to as the tails) and a polar, charged, hydrophillic head. This molecule is both hydrophobic and hydrophillic. Amphiphathic is the word we use to describe a molecule with both hydrophobic and hydrophillic properties. A main use for the phospholipid is in biological membranes, as shown in the image to the right.

Daily Challenge

Proteins, carbohydrates and nucleic acids have the ability to form complex polymers. Proteins and nucleic acids have their function is determined by the sequence of monomers. Phospholipids can spontaneously form sealed spheres that create an inside vs. an outside. All life functions rest upon the diversity of these chemicals. The basics of biochemistry that you have learned this week will be expanded upon throughout the coming weeks. You will see further examples of proteins, carbohydrates, nucleic acids and lipids.

Your goal is to start building mental models of what they are and how they interact. Draw out the structures, write out a full dehydration synthesis or hydrolysis. What factors cause proteins to fold? What would occur if you used saturated fatty acids in a phospholipid? What about non-saturated fatty acids? Take some time and really build an idea about each of these molecules, and appreciate the diversity.
After spending time, write in the discussion forum your thoughts and ideas about these biochemicals. (This is a free form discussion where you can write about proteins,carbohydrates and lipids. The focus is for you to express your understanding of the structure and function of the three biochemical categories, but you are also welcome to pose questions regarding what you do not understand about these chemicals.)
Link to Discussion Forum

Daily Newsletter: September 2, 2014 - Protein Folding

2014-09-02T12:56:00.003-04:00

Daily Newsletter

September 2, 2014 - Protein Folding

Yesterday's newsletter focused on amino acids, how these monomers are polymerized, and the importance of their R (functional) group. Today we are going to further examine the importance of functional groups by discussing how they help form the working shape of a protein by causing a chain of amino acids to fold.

The primary structure of a protein is a chain of amino acids. Due to how proteins form, one end of the chain will end in an amino group (N-terminus), which the other end will have a carboxyl group (C-terminus). In Between these two terminal points will be a wide variety of amino acids.
The side chains of neighboring amino acids will begin to interact. They could be pulled toward each other, be repelled, or have nothing happen. Remember that the functional groups can twist around the chiral (central) carbon of the amino acid, so repulsion may just force the side chains to opposite sides of the chain (remember, you are dealing with 3-D structures here). The amino groups and carboxyl groups, even though they are part of the back bone, also retain polarity. Thus they can also be involved in the folding.

These interactions start the formation of the secondary level of protein structure. The two most common types of secondary structures are the alpha helix and the beta pleated sheet. These two types of secondary structures will help explain how the amino acid side chains start the folding process.

The α-helix relies on neighboring amino acids. Through the polarity of amino and carboxyl groups, the backbone of the molecule begins to twist and hold due to electrostatic interaction (van der Waals forces). The image to the left is an example of an alpha helix. The alpha represents the direction of the twist, and you will learn more of the naming of these in organic chemistry. Notice that in the image there are yellow dashed lines. These represent hydrogen bonds (van der Waals forces) between amino acids. The green ribbon represents the backbone of the amino acid chain (amino-chiral-carboxyl connected to amino-chiral carboxyl and so on). So the interactions (notably from polar partially charged side chains has produced a twist in the primary structure.

In contrast, β-pleated sheet, shown on the right, has interactions between different regions of the primary structure. While only one amino acid chain is involved, in this case the chain is not twisting. Instead, neighboring regions become attracted to each other. Also, the electrostatic interaction is between amino and carboxyl groups, not R groups. Multiple regions can be brought together to form these sheets as indicated in the diagram below. In this diagram, the red arrows represent a portion of the primary structure that has begun to form β-pleates. Notice that six red arrows are arranged together. The purpose of this diagram is to show the placement of the β-pleated sheet. Consider a sheet. It is flat with two sides. Why do you think it would be important for a protein to fold in such a way as to create a relatively "flat" surface with two sides?

Notice that with the α-helix and β-pleated sheet we have altered the structure of the protein. It is no longer a linear chain, but has greater dimensionality. We have either turned the protein into a rope/cord (α-helix) or into a "plane" with two faces(β-pleated sheet). Now areas that were once distant have been brought closer together. Now side chains can start interacting with each other.
In the tertiary structure, different regions of the protein are brought into association. Electrostatic and hydrophobic interactions will force more conformational (structural) changes onto the protein. This will lead to a 3-dimensional structure. In the following diagram to the left, you can see an example of
a protein in primary and then tertiary structure.

Notice that the secondary structures are visible, but even these have been folded into each other.
The forces that govern this are found in the R (functional) groups of the amino acids. Hydrophobic areas cluster together. Positive and Negative charges attract, while like charges repel. Polar partially charged side chains further interact, either with each other, or with full charged. We also have a new interaction. The amino acid cystine contains a thiol (-SH). The thiols of two cystines can react to form a disulfide bond. This is a covalent bond. Question: Which is stronger individually, a covalent bond or a hydrogen bond (electrostatic interaction)? The disulfide bond is utilized to stabilize the 3-dimensional structure of the protein.

Many proteins are functional at the tertiary structure. Here you will see either globular or linear proteins (like collagen). There is a final level of structure. Some functional proteins are actually made up of multiple individual proteins. A great example of this is hemoglobin. The hemoglobin molecule, seen to the left, is composed of four individual proteins: 2 α-hemoglobin (red) and 2 β-hemoglobin (blue). The green structure is Heme, a prosthetic group that is used to hold oxygen, and is attached by electrostatic interactions (van der Waals forces) to the proteins.

NOTE: Some proteins are functional in the tertiary structure, but others are only functional when you have multiple individual proteins forming the quatrenary structure.
Denaturation: The protein is held together primarily through electrostatic interactions. What happens when a protein warms up? It starts to unfold. Why? The electrostatic interactions weaken as kinetic movement of the atoms increases. Acids and bases, with their charged H⁺ and OH^- also disrupt these electrostatic interactions. Secondary and tertiary structures begin to change conformation. Most notably, they unfold. To denature a protein is to unfold it....but....
What if you only apply a mild heat, let's say your muscles warm up due to exercise. What happens to the proteins? What happens to hemoglobin when it passes through a warm temperature? Your muscles are also metabolizing, and as we will see, produce acids. What does this do? So, is denaturation all or nothing?

The tertiary and quatrenary structures all have a specific electrochemical profile.
What happens if you add a charged particle/compound to a protein? What happens if I add a new positive charge? Answer: The protein will change shape (conformation).
What will this due to the function of the protein? It could actually active the protein, but it could also deactivate the protein. This will be a discussion a little later in the semester, but I want you to start thinking of the implications.

animation of hemoglobin t-r state transformation, made by en:User:BerserkerBen http://en.wikipedia.org/wiki/File:Hemoglobin_t-r_state_ani.gif

To the left is an animated image of hemoglobin. Notice that the configuration (shape) of the protein changes when Oxygen is bound (Oxy) and when no oxygen is bound (deoxy). MEME: When something binds to a protein, it changes shape. In the image, you can also see the various subunits (each a tertiary protein) with their associated Heme groups (heme groups are in red).

The folding of the protein is critical to its function. Even a small error in the primary structure can cause significant changes to the overall structure, and therefore changes to the function.

A well known condition with hemoglobin is Sickle Cell Anemia. The condition is based upon a mutation of the gene that codes for the β-hemoglobin. The switch from an Adenine to Thymine causes a change in the primary sequence of the protein. In wild type (non-mutant) β-hemoglobin, there is a glutamic acid (charged, negative) at position 6, but in Sickle Cell Anemia, the sixth position holds valine (non-polar). At low oxygen levels (such as in your tissues after exercise), the hemoglobin takes on an abnormal shape:


http://helicase.pbworks.com/f/1225738609/hemoglobins.GIF

The quaternary protein has now changed shape by opening up. Sickle hemoglobin has the ability to bond to other sickle hemoglobins. This is due to electrostatic interactions. The image blow shows the result of this interaction between sickle hemoglobin: clumping.

http://evolution.berkeley.edu/evosite/evo101/images/hemoglobin.gif

The clumping of sickle hemoglobin in low oxygen environments causes the distinctive change in shape seen in sickling red blood cells, and is all due to changing one amino acid in the primary structure. Below is an image showing normal vs. sickle red blood cells.

http://www.ama-cmeonline.com/pain_mgmt/pop_up/sickle_cell.htm

Daily Challenge:

Pick your challenge! Decide if you want to work on Discussion 1 or Discussion 2. Understand that you are responsible for considering both, and information from both could appear on future quizzes.

Discussion 1

Discussion 2

http://www.accessexcellence.org/RC/VL/GG/ecb/ecb_images/04_32_antibody.jpg

Daily Newsletter: September 1, 2014 - Polymerization and Amino Acids

2014-09-01T12:59:00.000-04:00

Daily Newsletter

September 1, 2014 - Polymerization and Amino Acids

This week is an introduction to biochemistry. It is critical that you recall how chemical bonds are formed, and more specifically the idea of covalent bonds (both polar and non-polar) and hydrogen bonds. Biochemistry is based on the fundamentals learned in first year chemistry, but to fully understand it, you will need organic chemistry. So your goal here is not to "master" biochemistry, but instead gain an appreciation and starting understanding of the chemistry of various biochemicals. The first step is to understand the concept of biopolymers and the process of polymerization.

Biopolymers

Proteins, Carbohydrates, Deoxyribonucleic Acid and Ribonucleic Acids are all biopolymers. The suffix bio- deals with life, so it is the root word polymer that we need to discuss. A polymer is a chain, or network of chains, composed of repeating chemical units. These chemical units, or monomers, are generally of the same chemical family, and have the ability to bind together to form larger structures (usually chains). For example, Glucose, a carbohydrate, can bind to Fructose (another carbohydrate) to form the disaccharide sucrose. In the mammalian liver, glucose can be bound into a repetitive chain of glucose monomers to create the polysaccharide Glycogen (poly=many, saccharide=sugar, so many sugars, or a polymer of sugars).

The monomers of biopolymers are linked through condensation reactions (dehydration synthesis) mediated by enzymes. For example, the biopolymerization of glucose into glycogen is handled by the enzyme Glycogen Synthase (note: there are some preliminary steps to get the monomer ready, but the polymerization reaction is handled by Glycogen Synthase).

(Basic Level Description) In dehydration synthesis (condensation), the removal of a hydroxyl from one monomer and a hydrogen from the second allows for the formation of a covalent bond.

OpenStax College. Dehydration Synthesis [Connexions Web site]. April 4, 2013.
Available at: http://cnx.org/content/m44397/latest/Figure_03_01_01.jpg

Amino Acids and Proteins

Proteins will be a reoccurring topic throughout the semester. They are one of the informational biopolymers. This means that they are composed of monomers (amino acids) that are linked together in specific sequences that are critical to their overall structure and function. [NOTE: It is important to understand the terms monomer and polymer, so make sure you have a good definition of these terms in your notebooks.] To understand proteins, we must first understand their monomeric unit, the Amino Acid.
Remember that all monomers will be chemically similar. In the case of the amino acid, the base molecule of Amino-Chiral Carbon-Carboxyl is the same. The difference in the amino acids comes with the side chain. These functional groups give each amino acid its unique identity and function. The twenty amino acids that are used in natural proteins can be found in the following link: Amino Acid Diagram. Notice that there are four general classes of amino acids with different chemical properties based upon the functional group. NOTE: In the diagram to the right, R represents the radical group. This is the funcational group or side chain. The distinctive characteristics of an amino acid are determined by the R group.

This linking of amino acids through peptide bonds will create the primary structure of a protein. All of the remaining levels of protein structure will result from interactions between functional groups on the amino acids. Local interactions induces folding into the secondary structures, which will result in other amino acids coming into close contact. This results in a tertiary structure. Finally, you will get a quaternary structure when multiple individual folded peptide chains come together. Remember that Van der Waals forces (including hydrogen bonds), covalent bonds (disulfide bridges), and hydrophobic interactions will all induce folding. Because of this, environmental factors (such as heat or pH) can influence the folding pattern and shape of a protein.

Daily Challenge

In the above diagram of the dipeptide (two amino acids connected by a peptide bond), notice how the the R (functional group) is on the same "side" of the molecule.

When you get to organic chemistry, you will discover that there can be rotation around a carbon atom, especial the chiral carbon at the center of each amino acid. What this means is that the R groups can rotate away from each other toward each other. Consider the different types of amino acids, and most notably their possible interactions.

Your challenge today is to discuss how the different types of amino acids (their functional groups) can react with each other. Stay focused on the "types" and not individual amino acids at this time. To focus your discussion, what would happen if you had a chain of all one type? What would happen if you alternated types? How would you form a section of the protein to interact with a hydrophobic environment and a hydrophilic environment at the same time?

Daily Newsletter: August 29, 2014 - Characteristics of Life

2014-08-29T15:29:00.000-04:00

Daily Newsletter
August 29, 2014

__________________________________________________

Characteristics of Life

In order to have a meaningful discussion about living systems, we must first identify that which we will consider "living." To do this, we identify characteristics that will allow us to differentiate between living and non-living objects. To be effective, these characteristics must be see in all examples of life.

As a first principle, we will start with the cell theory: All living organisms are composed of cells, and all cells come from pre-existing cells. Thus, the cell is the basic unit of life. One of our goals this semester is to gain an understanding of the cell, and build a working model of how a cell functions. As a frame four this semester long discussion, let's first look at how the characteristics of life are revealed in a cell.

Homeostasis: i.e., the same state. This refers to the ability of a cell to maintain a constant 'internal' environment. The environment around the cell can change (e.g., hot to cold, low salt to high salt), but the cell it self remains stable. [Caveat: All organisms have a range of environmental conditions where they can live; if you exceed the range, then the cell can not maintain homeostasis]. We shall see that the cell membrane is the defining structure of all cells, as it creates a boundary (inside vs. outside). It is from this basic idea, and how we move things across the membrane, that we shall build the concept of homeostasis.

A corollary characteristic is adaptation. Cells can change in response to the environment. For example, bacterial cells can change the phospholipids in their cell membrane as temperature changes. One way this is done is by changing the amount of saturated fatty acids used in making phospholipids.
In order to adapt, you need to know how the environment changed. This is done through systems of cellular receptors. Stimulus (environmental change) ---> Receptor -----> Adaptation. So cells also have the ability to Respond to Stimuli. These stimuli could be physical factors (temperature or pH), or they could be chemical. The thing to remember is that cells (and all life) have some type of Stimulus-Response (Receptor or Communication) system.

Growth: It is easy to see a human growing from infant, through adolescence, to adulthood. But what about cells? We could look for the growth in size, but that can be difficult when looking at a single cell. Instead for cells, we look at Cell Number (population size) and/or Biomass (e.g., the amount of carbon or maybe the dry weight of the population). Growth is based on two other characteristics of life.

Metabolism: i.e., all chemical reactions in the body. Often down played to just energy acquisition, metabolism also involves the acquisition of Carbon (needed for all biochemical compounds), other essential elements (like nitrogen), and the building of new biomolecules. As cells make more biomolecules, they grow, and eventually divide.
Reproduction: reproduction is essentially the creation of new individual organisms, and is easily seen when a baby is born. But reproduction has its basis in cellular action. A cell, when given the correct stimulus, can divide. Possibly it has grown too large, or it may have received a chemical signal to divide. In either case, a cell will divide into two daughter cells. Terms that will come up with reproduction are: binary fision, mitosis, meiosis, and cytokineses.

You may remember from other courses that Organization is often given as a characteristic. Instead of talking about tissues and organs, I would remind you that living organisms are composed of cells. If you have active, working cells, then you are living. Doesn't matter if it is one cell or a million.

Daily Newsletter: August 27,2014 - Scientific Thought in Biology

2014-08-27T12:42:00.003-04:00

Daily Newsletter

August 27,2014

Scientific Thought in Biology

The goal today is to start to look at the questions: what is science? how do we do science?

Science is more than a collection of facts. Facts are important. We are constantly uncovering new facts. But it is the analysis and incorporation of these facts into historic body of knowledge that makes up the foundation of science. Science is dynamic, and we are ever increasing our knowledge base.

Let's look at a slightly different question: What is the goal of science?

Take a moment and think about it.

Why do people try to "figure" things out?

Why do they "do" experiments?

One of my favorite phrases in answer to this question is that science provids us the power to predict and control natural phenomena. Such as predicting storms, identifying diseases, better crop management, and disease treatment to list just a few ways we predict and control natural phenomena. Consider, we predict changes in the flu, and then control flu outbreaks through flu vaccination.

If you start looking into the Philosophy of Science you will find a number of different definitions, but for us, the above definition starts the discussion.

So how do we go about science? Do we just propose an idea on how it works and leave it? No! We always want to TEST our ideas, and see if they provide a workable model. Our scientific method is based on the Hypetheticodeductive Model of Reasoning. Instead of naming the "steps" of the scientific method, I want you to instead break it down and really think about what it means.

First off, look at the word Hypetheticodeductive. It is constructed of two words: Hypothesis and Deductive. These are the two critical features, but what do they mean. So, what is a hypothesis, and what does it mean to be deductive?

Consider the following:

Critical Concepts

Hypothesis - A proposed explanation of a phenomena.

You may remember this definition from other science classes. It is a good place to start, but for a strong hypothesis, there are characteristics you must take into account. The following characteristic are not all inclusive, but they are important ones to begin considering.

A hypothesis must be testable, and conceivably falsifiable.

You must be able to conceive of a way to legitimately test the hypothesis.
There must be conditions in which the test could reveal a negative/false result.
If there is no possible way to generate a negative result, then you do not have a scientific hypothesis (we did not find any evidence of ghosts, but that does not mean you don't have ghosts).

Succinctness - A hypothesis should have few underlying assumptions. The more assumptions, the weaker the hypothesis (also called parsimony).

Discussion

Dr. Ignaz Semmelweis is a figure rarely discussed in general biology, but his work as one of the founders of Infection Control is important. His work also stands as an incredible example of the hypethetico-deductive model at work:

Read the following websites/articles. It is suggested that you read them in order.

In the discussion forum for today, describe how Dr. Semmelweis' work with puerperal fever demonstrate the scientific method at work. Look at the hypotheses he generated, and consider whether these were strong or weak? Does the data shown in the fourth link provide support to Dr. Semmelweis' final solution? Did Dr. Semmelweis have the correct cause, or were there other discovers that ultimately explain what was happening in the First Ward? Discuss the concept of an Agent of Change; how did Dr. Semmelweis fail as an agent of change, and consider how you would stand as an agent of change.

NOTE: Don't answer these as individual questions. Consider and build a response.

Microbiology Daily Newsletter February 20, 2014 - Transcription Termination

2014-02-20T17:40:00.002-05:00

Microbiology Daily Newsletter

February 20, 2014 - Transcription Termination

Transcription Termination

The elongation phase of transcription (polymerization of RNA) occurs the same in all domains, working via base complementarity. Termination, like initiation, is different. In bacteria, transcription termination occurs either through an intrinsic termination system (Rho-Independent) or through the use of a Rho (ρ) factor (Rho-Dependent).

The Intrinsic System (Rho-Independent)

After a the coding region of the gene, or the final coding region of an operon, there will be an inverted repeat of nucleotides on the DNA. Following this repeat will be a series of six adenine nucleotides. When this region is translated, the inverted repeat (usually rich in G-C) will anneal, creating a hairpin loop structure. The six adenine nuclotides are transcribed into uracil.

A protein associated with the RNA polymerase, nusA, catches the hairpin loop structure, and holds it. This stalls transcription in the region where Uracil has been transcribed. A region of A-U in a DNA-RNA duplex (i.e., strand of RNA bound to a complementary DNA strand) is very weak (it is a major weak point). This naturally dissociates from the DNA strand, and is released by the RNA polymerase.

Summary: The intrinsic system relies on the stalling of a hairpin loop at the end of the transcript, and a region of A-U, to terminate transcription.

The Rho-Dependent System

The ρ-Factor is an ATP-dependent hexameric helicase, and binds to a cytosine rich region of RNA >70 bases upstream from the termination site. This cytosine dominant region is known as the Rho Utilization Site (abbreviated rut). Once bound, the ρ-Factor moves up the RNA strand toward the 3' end (e.g., where the DNA-RNA duplex is found). RNA Polymerase pauses at the termination site, and during this time, the ρ-Factor comes catches up to the transcription fork (DNA-RNA duplex), and unwinds it. Transcription ends.

Comparison between the two termination systems


Comparision of intrinsic termination and Rho-mediated termination. Greive S.J. and P.H. von Hippel. (2005) Thinking quantitatively about transcriptional regulation. Nat Rev Mol Cell Biol 6:221-32.

Eukaryotic Transcription

To refresh your memory regarding Eukaryotic transcription, you may want to read over these articles:

Daily Challenge

Your goal today is to discuss the differences similarities and differences between bacterial and eukaryotic transcription, specifically initiation and termination. Read DNA Transcription in case you need a refresher.
REMEMBER to discuss both similarities and differences. While doing this, consider evolutionary differences.

Microbiology Daily Newsletter February 18, 2014 - Transcription Initiation

2014-02-19T13:01:00.000-05:00

Microbiology Daily Newsletter

February 18, 2014 - Transcription Initiation

Hopefully you remember the basic idea of a gene and the mechanism of transcription as it applies to eukaryotic cells.

For bacterial transcription, we will first look at the promoter. You may recall the TATA box with eukaryotic promoters. In bacteria, we find the Pribnow-Schaller box, commonly just referred to as the Pribnow box. The Pribnow-Schaller box is a six nucleotide sequence TATAAT. QUESTION: Why six nucleotides?

With eukaryotic promoters, you may recall that the TATA box is not the only recognition sequence needed. This is also true of prokaryotic promoters. The Pribnow-Schaller box is found at -10 from the start of the gene. The complete promoter also contains a -35 recognition sequence, and is also comprised of six nucleotides. The -35 promoter element usually has a sequence of TTGACA, but note that this can vary among different bacterial taxa (usually at the class or family level taxa). NOTE: The -10/-35 promoter is used for normal house keeping genes. We will see some variation shortly.

The prokaryotic RNA Polymerase catalyzes the reaction of both coding and non-coding RNA, unlike eukaryotes that have job specific RNA polymerases (for example, you may remember that RNA-Pol I forms the 45S pre-rRNA, while RNA-Pol III forms tRNAs). The prokaryote RNA Polymerase Complex is a holoenzyme composed of RNA Polymerase and a Sigma (σ)factor. The eukaryotic RNA Polymerase complex has a specific RNA polymerase and a variety of initiation factors.

Below is an example of the bacterial transcription initiation complex. Note that you are seeing RNA Polymerase with 2 different σ factors, and that the different sigma factors have slightly different promoter recognition sites. When they bind, they move from a closed complex to an open complex; the open complex gaining its name from the opening of the DNA helix.

Add cFrom: Bush M , and Dixon R Microbiol. Mol. Biol. Rev. 2012;76:497-529. Initiation of transcription by the RNAP-σ70 (A) and RNAP-σ54 (B) holoenzymes. The σ70 factor directs the binding of polymerase to the consensus −10 (TATAAT) and −35 (TTGACA) sequences to form an energetically unfavorable closed complex (CC) that is readily converted into an open complex (OC) to initiate transcription. In contrast, the σ54 factor directs the binding of RNAP to conserved −12 (TGC) and −24 (GG) promoter elements that are part of the wider consensus sequence YTGGCACGrNNNTTGCW (where uppercase type indicates highly conserved residues, lowercase type indicates weakly conserved residues, N is nonconserved, Y is pyrimidines, R is purines, and W is A or T) (10). This forms an energetically favorable CC that rarely isomerizes into the OC. In order to form the transcription “bubble,” a specialized activator (a bacterial enhancer binding protein [bEBP]) must bind and use the energy from ATP hydrolysis to remodel the holoenzyme. aption

Once the RNA Polymerase-σ factor Holoenzyme is bound in the open complex form, transcription can proceed to the elongation phase. The next issue specific for prokaryotic transcription will be termination.

Daily Challenge

Sigma factors play a critical role in coordinating bacterial cell physiological states. Below is a list of common sigma factors.

Sigma Factor	Gene	Function
s⁷⁰	RpoD	Primary s factor, Housekeeping
s¹⁹	FecI	Regulates fec gene for iron transport
s²⁴	RpoE	Extreme heat stress
s²⁸	RpoF	Flagellar genes
s³²	RpoH	Heat shock
s³⁸	RpoS	Starvation/Stationary phase

Along with these can be found anti-sigma factors that block the function of expressed sigma factors (a form of regulation). Discuss the role of sigma factors as means of global gene regulation. Why is having a sigma factor system beneficial to bacteria? How does this differ from eukaryotic promoters?

Microbiology Daily Newsletter February 17, 2014 - Bacterial Genetics

2014-02-17T14:51:00.000-05:00

Microbiology Daily Newsletter

February 17, 2014 - Bacterial Genetics

At this point, all students should be able to provide a solid description of Replication, Transcription and Translation. Therefore, we will not concern ourselves with the basics of these processes, but instead focus on how Bacteria differ from Eukarya in these three processes. As we move though this week, we will also begin our discussion on genetic regulations.

The following topics in genetics you should have a strong familiarity with. If you don't then it is time to brush up on your genetics.

Be able to discuss replication, transcription and translation.
Be able to describe in detail the mechanisms of replication, transcription and translation.
Be able to discuss the concept of a genome, and what constitutes a genome.
Be able to discuss supercoiling of DNA and the use of topoisomerases.
Be able to discuss how DNA analysis is done, and why.
Be able to discuss how researchers analyze the whole genome, and why it is important.
Be able to discuss all of the different types of RNA.
Be able to discuss post-translational modification of proteins.
Be able to describe open reading frames, paralogs, orthologs and DNA alignments.
Be able to discuss various mechanisms of gene regulation and epigenetics.

Bacterial DNA Replication

The genetic process of replication is fundamental to all living systems, and the basic mechanism is the same: DNA is unwound and opened, each parental strand acts as a template upon which a newly synthesized strand is based; the resulting daughter molecules are semi-conservative, being formed of one template (old) strand and one new strand. You should be intimately familiar with this process.

So, what is different between bacterial replication and eukaryotic replication?
Remember that the nucleoid (genophore) of the bacteria is a single circular molecule that is vastly smaller than eukaryotic chromosomes. Unlike the chromosome, which can have multiple origins of replication, bacteria will generally only have 1 origin of replication (Ori).

Eukaryotic chromosomes are linear, and so you keep replicating til you get to the end (remember, there is a problem with the ends potentially degrading...remember telomerase?). Bacterial DNA is circular, so there is no end. Instead, you find a terminus, or termination point (Ter).

As with all DNA replication, you will get two replication forks on either side of the origin of replication, these will then meet at the terminus. All the while, the DNA is anchored to the cell membrane, and it is the movement of the cell membrane that will separate the two daughter molecules.

The process begins when DnaA binds to the Ori. DnaA, and the specific sequence at Ori, are genera/species specific. The most highly studied system is Escherichia coli, where a repeat of 5' - TTATCCACA - 3' is used as the binding sequence for DnaA.

DnaA enzymes needed for replication including Helicase, DnaB, and DnaC. DnaB anchors at what will become replication forks, and aids in the binding of primase.

Single-stranded binding proteins and DNA gyrase will be needed to prevent re-annealing and positive supercoiling respectively. DNA polymerase III holozyme will be the primary work horse of the elongation phase.

Termination is interesting, as the two replication forks are forced to meet in the Ter region, by a process referred to as the replication fork trap. The two daughter strands are joined as interlocking rings in the terminus (catenane, or mechanically linked molecular architecture). DNA Topoisomerase IV will be the critical player in unlinking the daughter molecules, and will need DNA gyrase to assist.

From: Charvin G et al. PNAS 2003;100:9820-9825. Configurations of a circular replicating DNA. (a) Model for in vivo strand separation: the progression of the replication complex leads to the formation of (+) supercoils (L-nodes) in front of the replication fork and R-precatenanes behind. Topo IV removes (+) supercoils and gyrase generates (–) supercoils so that, under the action of both enzymes, the chirality of the precatenanes is inverted. Topo IV may then unlink the molecules by removing the L-catenanes. (b) Model for in vitro decatenation by Topo IV: R-catenated plasmids may form L-supercoils that are removed by Topo IV with a high rate [as observed in our experiments and some bulk assays (21)]. However, the removal of the last few links is slow , because Topo IV will relax only a rare fluctuation of an R-node to an obtuse angle that is similar to its preferred substrate: an L-node with acute angle (see Fig. 1b).

Daily Challenge

Review DNA replication as it pertains to bacteria, and consider, how would you move from the replication of a circular DNA molecule to a linear molecule? What would need to change? Initiation? Elongation? Termination? How different are each of these process from eukaryotic replication?

BOLO Microbiology Daily Newsletter February 4, 2014 - Metabolism & Growth

2014-02-04T13:58:00.000-05:00

Microbiology Daily Newsletter

February 4, 2014 - Metabolism & Growth

Metabolism and Growth are both essential characteristics of life, and are intimately related to each other. You may recall that living systems require CHONPS (carbon, hydrogen, oxygen, nitrogen, phosphorous, and sulfur) to make biomolecules. In addition, we need minerals, trace minerals, and energy in the form of reducing potential (i.e., electrons). The acquisition of carbon and energy are the primary focus of central metabolism (glycolysis, citric acid cycle, etc...) and photosynthesis. But what about the other needs?

Amino acids and nucleic acids both need nitrogen. Nucleic acids need phosphorus in the form of phosphate groups. Sulfur is needed to form disulfide bridges and a variety of thiols. Phosphates become a prime regulator of protein function, and ions are needed to establish membrane potentials. All of these are needed to increase the biomass (growth) of cells.

In looking at microorganisms you need to become accustomed to the metabolic needs and diversity of cells. Though less than 1% of bacteria are culturable, these organisms have helped us understand the dynamic relationship between nutrition, metabolism and growth. They have also provided researchers with known genes that can be used to understand potential functions from genetic analysis.

Today, your goal is to understand how variations in growth conditions can affect bacterial growth, both in terms of biomass accumulation and increases in population size (binary fission).

Heterotrophy vs Autotrophy

These two terms describe the acquisition of carbon. Heterotrophy is used to describe organisms that rely on organic carbon as building block. Autotrophy describes organisms that have the ability to fix carbon dioxide into an organic compound (carbon fixation means adding a CO₂ onto an existing organic carbon backbone; this makes the carbon biologically available).

Phototrophy vs Chemotrophy

These two terms describe the acquisition of energy in the form of reducing power. Phototrophs have the ability to utilize photons to generate high energy electrons that can be used as reducing potential. Chemotrophs utilize reduced compounds for reducing potential (i.e., energy).

The concept of chemotrophs can be further divided in to chemoorganotrophy, in which cells utilize reduced organic compounds for energy, and chemolithotrophy, where cells utilize reduced inorganic compounds for energy.

Nitrogen

Molecular nitrogen is the most abundant gas in our atmosphere, but it is not very accessible by most biological organisms. For nitrogen to become available, it needs to be reduced. Atmospherically this is accomplished by electrical activity, but far more becomes available through biological means. Nitrogen fixation is an important chemical pathway, and is the first step in the nitrogen cycle (the movement of nitrogen through ecosystems). The diagram below provides an explanation of nitrogen fixation:

Plant Biotechnology Handbook by Niir Board.

Note the amount of redox reactions needed, and the requirement for Mo (Molybdenum) as a cofactor for the enzyme nitrogenase (looking at the nitrogenase cycle, how is molybdenum used?). Notice also that Magnesium (Mg) is also needed during the reduction of molecular nitrogen. Nitrogen has a number of biologically active states: including NH₃, NH₄, NO₃, NO₂, and NO. The movement of nitrogen through living systems is shown in the diagram below:

Nitrogen fixation.http://archive.bio.ed.ac.uk/jdeacon/microbes/nitrogen.htm

Can humans use raw NH₃? How about bacteria? Can all bacteria use NH₃, or only some? These questions come to the heart of today's topic. All bacteria need nitrogen, but they differ in how they acquire nitrogen. Some can only harvest nitrogen from organic compounds, while others can make use of some of these other compounds. Bacteria also have to give off nitrogen as waste, but how do they do it? We convert ammonia to urea, but do bacteria need to make urea? This also brings up another point about living in a community of different types of bacteria: the waste of one cell, may be the food for another cell.

Daily Challenge

What determines the metabolic diversity of a bacterial species? What other conditions do you need to consider when growing bacteria? How does central metabolism provide raw materials needed for growth?

BOLO Biology Newsletter Archive

Introduction to Eukaryotic Gene Regulation

lac operon

Challenge

Challenge

Introduction to Gene Regulation

Study Skills

Suggested Reading

Daily Challenge

Study Skills

Amino Acids and Polymerization

Protein Self-Assemble (Folding)

Challenge:

Challenge 1

Challenge 2

Translation

Initiation of Translation

Elongation

Termination

Daily Challenge

Optional Challenge: Genomics and Proteomics

Optional Video

Codons, Anticodons & Amino Acids

Challenge

Group Projects: History of Biology Timelines

Update: Summer 2015

Special Newsletter Gibbs Free Energy and Transition State Energy (Reposting)

Special NewsletterGibbs Free Energy and Transition State Energy

Take home message:

Daily Newsletter September 24, 2014 Redox and Coupled Reactions (Reposting)

Daily Newsletter

September 24, 2014

Redox and Coupled Reactions

Daily Challenge

Daily Newsletter September 23, 2014 Adenosine Triphosphate (Reposting)

Daily Newsletter

September 23, 2014 Adenosine Triphosphate

Chemist vs. Biologist

Daily Challenge

Reference

Special Newsletter: Gibbs Free Energy and Transition State Energy

Special NewsletterGibbs Free Energy and Transition State Energy

Take home message:

Daily Newsletter: September 10, 2014 - Codons, Anticodons & Amino Acids

Daily Newsletter

September 10, 2014

Codons, Anticodons & Amino Acids

Daily Challenge

Daily Newsletter: September 8, 2014 - Ground Rules for Gene Expression

Daily Newsletter

September 8, 2014

Ground Rules for Gene Expression

(AKA Central Dogma)

Daily Challenge

Daily Newsletter: September 5, 2014 - DNA Replication

Daily Newsletter

September 5, 2014 - DNA Replication

Replication

Daily Challenge

Daily Newsletter: September 4, 2014 - Nucleotides

Daily Newsletter

September 4, 2014 Nucleotides

Nucleic Acids

Base Complementarity:

Directionality

Daily Challenge

Daily Newsletter: September 6, 2013 - Carbohydrates and Lipids

Daily Newsletter

September 6, 2013 Carbohydrates and Lipids

Carbohydrates

Lipids

Daily Challenge

Daily Newsletter: September 2, 2014 - Protein Folding

Daily Newsletter

September 2, 2014 - Protein Folding

Daily Challenge:

Discussion 1

Discussion 2

Daily Newsletter: September 1, 2014 - Polymerization and Amino Acids

Daily Newsletter

Special Newsletter
Gibbs Free Energy and Transition State Energy

Special Newsletter
Gibbs Free Energy and Transition State Energy

Daily Newsletter
August 29, 2014