Thursday, January 15, 2015

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

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=ΔHTΔS

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)

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!
Nicotine Adenine Dinucleotide
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)

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).
Protein Phsophorylation
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). Myosin Cross Bridge (Moving Myosin Heads Bind to Actin)
.

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

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: September 24, 11:55pm.
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

Friday, September 19, 2014

Special Newsletter: Gibbs Free Energy and Transition State Energy

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=ΔHTΔS

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.

Tuesday, September 9, 2014

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

Site LogoDaily 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.
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?

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

Monday, September 8, 2014

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

Site LogoDaily 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 Major Groovemolecule. 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. Major Groove with Initation FactorThe 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. Gene 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.

tRNAThe 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 3rd 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.
Genetic Code Algorithm
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.
Genetic Code

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

Friday, September 5, 2014

Daily Newsletter: September 5, 2014 - DNA Replication

Site LogoDaily 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.
DNADNA 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.

DNA Polymerization
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.Phosphodiester Bond Formation 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.DNA ReplicationThe 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