Daily Newsletter September 30, 2013 - Glycolysis steps 1-5

Daily Newsletter

September 30, 2013 - Glycolysis steps 1-5

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Today we start looking at the metabolic pathway of glycolysis. Recall that in catabolism we are harvesting carbon (heterotroph) and energy (chemotroph). Moment by moment, cells are having to determine whether they have a need for carbon or energy. Intermediates from our catabolic pathways provide precursors for other biochemicals, but they also provide energy in the form of reducing potential.  Reducing potential can be used for anabolic reactions, but will also be used to generate ATP.  Remember:  catabolic pathways will yield carbon precursors, reducing power (potential), and ATP.

With digital technology, you can go on line and pull down images of the glycolytic pathway. Nearly every biology textbook has these images. Your goal this weak is NOT to memorize these metabolic reactions. Your goal is to understand what is happening. If you have an idea of how to read a metabolic pathway, then you can look at any pathway given to you and have an understanding of what is happening.  To do this, you need become familiar with enzyme names and common reactions.  As you take organic chemistry, your ability to read metabolic pathways will improve, but remember you first need a foundation (this is your foundation).

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Glycolysis

Glycolysis describes the splitting of glucose into two three-carbon Pyrvate molecules. This pathway consists of 10 reactions that carry out this splitting by inducing specific changes into the molecule. The first five steps are classified as preparatory, or energy consumptive. In these steps, we are preparing the molecule of glucose for the first split.

Glucose is chemical stable. Glucose does not spontaneously explode or degrade. Chemical stability also implies that it does not react easily. So, we need to make it more reactive. We also need to get it into the correct configuration for splitting. That is the goal of the first four steps.

In step 1, we use the enzyme hexokinase (specifically Glucokinase). The root word here is kinase. A kinase is an enzyme that adds a phosphate group to a molecule. In this case, hexokinase is an enzyme that adds a phosphate group to a six carbon sugar, in this case glucose. You will notice in the above picture that the enzyme is referred to as glucokinase. Glucokinase is a specific hexokinase (Hexokinase IV), and is found in specific mammalian cells such as the intestines, liver, pancreas, and brain.(Recall, the hydrolysis of the terminal phosphate of ATP is coupled with the covalent bonding of the phosphate to the substrate).

Hexokinase is generally attached to the glucose carrier found in the cell membrane. When glucose is brought into the cell, hexokinase adds a phosphate group to the sixth carbon. Glucose 6-P refers to a glucose molecule with a phosphate on the sixth carbon. Glucose and Glucose 6-P are different molecules, with their own concentration gradients.  By doing this, we remove glucose from the inside of the cell (it has become Glucose 6-P).  As such, there is always a glucose concentration gradient (outside high, inside low).

In order to carry out this phosphorylation, we use ATP. ATP transfers the terminal phosphate to the sixth carbon of glucose via a coupled reaction. (enzymatically, how would this happen? Would you need both ATP and Glucose in the active site?)

Why do we need to phosphorylate glucose? This is an important question, and something you should ask for every metabolic reaction. Why do we need to do it? What is the end product? You should also ask yourself questions about the enzyme.

• Why do you phosphorylate glucose?
• Glucose is stable, so the addition of phosphate with its -2 charge causes an electrical instability in the molecule.
• Glucose 6P is more reactive than Glucose.
• Glucose 6P can not leave the cell through the Glucose Carrier (they are different molecules now).
• Glucose 6P does not interfere with the concentration gradient of Glucose (they are different molecules, each with a concentration gradient).
• Glucose then remains high on the outside of the cell, but almost zero inside of the cell (incredibly strong concentration gradient).

• What about the enzyme?

• Is it regulated?
• Unidentified allosteric regulation.
• What is the structure?
• 465 amino acids
• What does the active site look like?
• Active site fits Glucose and ATP.
• What about the activity?

This first reaction has a ΔG^o' of approximately -4 kcal/mol. This indicates a spontaneous reaction, but as we have discussed above, the reaction is not spontaneous, so why such a large negative ΔG^o'? Coupled Reactions. You are seeing the overall reaction in the above image, but there are two reactions taking place: removal of Phosphate from ATP, and the addition of Phosphate to Glucose. Remember that in a lab, ATP yeilds a large release of energy. In biology, this energy is coupled to the building of the phosphate bond to glucose. ATP helps to drive the reaction of the stable glucose molecule.

In the second reaction of glycolysis, the enzyme phosphoglucose isomerase (Glucose-6-phosphate isomerase) causes the glucose molecule to rearrange slightly into the the hexose isomer fructose. Glucose and Fructose have the same chemical formula, but different chemical structures. Notice that glucose forms a six sided ring, with the sixth carbon outside of the ring. Fructose forms a five sided ring, with the 1^st and 6^th carbons outside of the ring structure. Why is that important?

The ΔG^o' for this reaction is +0.4 kcal/mol; so in a lab it is not expected to proceed at significant levels. Enzymes though allow for this reaction to proceed at significant rates. Of course, a build up of fructose-6-phosphate would disrupt the dynamic equilibrium we want, so we move this product to become the substrate of the next reaction.

Reaction 3 is catalyzed by phosphofructokinase. This enzyme will add a phosphate group onto the 1^st carbon, and the reaction has a ΔG^o' of -3.4 kcal/mol. Notice that the free energy is less than in the first reaction. Why? Think about what is happening; we are adding a second phosphate to a molecule that already has a phosphate. We are adding a large -2 charge to a molecule that already has a large -2 charge. This is not energetically favorable, so more of the energy from ATP is needed to get this reaction to go.

Now look at the resulting molecule: Fructose 1,6-bis phosphate. There are two carbons sticking out of the ring structure. In the image, you are seeing them on the same plane (above the ring). The phosphates have a -2 charge. What are they doing to the molecule? (Hint: Molecular Tension). To the right is another image of fructose-1,6,-bisphophate to help you visualize the resulting tension in this molecule. NOTE: bis is an archaic prefix to indicate two or second instance of something. In modern English, we usually us bi-, but organic chemistry (and more importantly IUPAC) still uses the bis- prefix.

In the fourth reaction, we see the splitting of the glucose molecule. The molecular tension is focused on the covalent bond between the 3^rd and 4^th carbon. Aldolase is the enzyme that catalyzes this reaction. This reaction deals with some specific organic chemistry, so we will hold off on the full discussion at this time. The goal of the reaction is the splitting of glucose into two 3-carbon isomers: Glyceraldehyde 3-P and Dihydroxyacetone Phosphate (DHAP). The problem here is that DHAP is a metabolic dead in in our current pathways. We do no want to loose the energy and carbon held in DHAP, so we need to convert it into something useful. That is the goal of reaction 5.

NOTE: the ΔG^o' of reaction 4 is generally reported at +5.7 kcal/mol. Remember that these numbers are from isolated reactions. Covalent bonds are strong and stable; even with the molecular tension of 2 phosphate groups, the bond between carbon 3 and 4 does not want to break. That is where the enzyme aldolase comes in. If you remember from lecture, we talked about what can happen at an active site. One of the options was inducing physical stress and charges. What would happen if you had complementary positive charges for the phosphate pulling away from the fructose? What would happen if you physically pulled these regions apart? Would it be easier to break the covalent bond?
In reaction 5, we convert DHAP into a more usable form, namely Glyceraldehyde 3-Phosphate (G3P is a common acronym). The enzyme that carries out this reaction is shown in the above diagraph: Triosphosphate Isomerase.

At the end of these first five steps, two phosphates have been added to a hexose in order to destabilize the sugar and cause it to break in a very specific manner (between carbon 3 and 4). The result is that we have two 3-carbon sugars: G3P. Notice the oxidation state of the molecule. There are a number of hydrogens attached directly to carbon. There is energy that we can harvest in the form of reducing potential. Tomorrow we will continue with steps 6-10.

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Daily Challenge

In your own words, describe steps 1-5 of glycolysis. Discuss how the enzymes make the reactions possible, including how they can couple reactions (this is important for any reaction with ATP). Discuss how the stepwise change in the molecule allows for the localization of the bond that needs to be broken. Why don't we just break that bond immediately? Remember, this will become part of your next milestone paper.

Due October 2, 2013 at 11:55pm.

Daily Newsletter: September 25, 2013 - Enzymes

Daily Newsletter

September 25, 2013 Enzymes

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Enzymes are the workhorses of the cell. They catalyze reactions, meaning they decrease reaction activation energy. It is critical to learn how enzymes function. To start with, here are some things you need to remember.

• First thing to remember, Enzymes are Proteins!
  • So they are constructed on ribosomes.
  • Their structure is determined by electrostatic interactions.
  • Their shape can change when things bind to it.
  • They can be denatured.
• Second, they act as catalysts.
  • They lower activation energy by bringing molecules together in the correct alignment, and can induce molecular tension to cause the reaction.
  • Though their shape may change during the chemical reaction, the enzyme is left essential unchanged at the end of the reaction.

Enzymes work by binding the substrate of the reaction, and then inducing molecular tension. Remember, when something binds to a protein, the electrostatic interactions around the protein change, resulting in a conformational (shape) change in the protein. It is this shape change that will induce molecular tension. Molecular tension could be due to physical strain, electrostatic interaction, or even the formation of temporary chemical bonds.

Every enzyme has an active site. This is the place where the substrate(s) will bind to the enzyme. The active site must have a shape that loosely fits the substrate, and the electrochemical pattern of the active site must compliment the electrochemical pattern of the substrate. When they bind, you get an Enzyme Substrate complex. Below is a basic cartoon about the process:

This enzyme-substrate complex is critical, and it is likely that you will see the development of an intermediate during the process. As the substrate "binds" to the active site, the overall enzyme will change shape. This conformational change is part of how the enzyme lowers the activation energy of the reaction. In the case of a synthesis, the enzyme will force the two compounds into close association, while in a break-down reaction, the enzyme may appear to bend the molecule at a breaking point. Here is a quick video that shows the brief conformational change that helps to induce the reaction: http://youtu.be/V4OPO6JQLOE



Conformational changes alone are not the only part in inducing a reaction. You will also find that proteins can have prosthetic groups that aid them in their action. For example, you can a Heme group with Iron that can hold Oxygen in red blood cells. Some digestive enzymes use Chromium to help in their action. Many metabolic pathways will contain Electron Carriers needed for redox reactions.

Enzyme Regulation
Cells are filled with enzymes, and they are constantly working to maintain cellular homeostasis. Some of the enzymes though are produced in an inactive form. We need them, but not always. We are also able, though chemical signals, to turn off some enzymes. Last week, we looked at signal transduction, converting a chemical signal into a change in cellular physiology. We saw the action of protein kinases and other primary and secondary signals. One purpose of the protein kinases was to alter the activity of proteins in the cell.

Beyond an active site, many enzymes have an allosteric site (regulatory site). This is located away from the active site. When the regulatory compound "binds" to the allosteric site, the enzyme changes shape. Most critically, the enzyme alters the shape of the active site. An allosteric activator is a signal that opens the active site, while an inhibitor closes the active site. The image to the right shows an enzyme in the active and inactive state. Notice that the active site changes when the allosteric activator is bound to the enzyme. In the case of this enzyme, there are two different ways to regulate: Phosphorylation, which would have occured due to a chemical signal such as cAMP, and the binding of ATP at an allosteric site.

It is not uncommon to have multiple ways to regulate an enzyme, and some enzymes that can be activated and deactivated as needed. A good example of this is with the enzyme Phosphofructokinase, a critical enzyme in glycolysis. This enzyme catalyzes an energy consumptive reaction that is a "no turning back" point in glycolysis.

Before we talk about phosphofructokinase, we need to talk a little of why we regulate proteins. Two memes I want you to remember: Cells are masters at energy conservation & Cell do not carry out unneccessary reactions. Both concepts are related, and they will help you understand why proteins are regulated. While there are energy consumptive pathways, the main reason we regulate proteins and pathways is due to products. Remember our discussion of equilibrium. We need to use products (products must become the next substrate). If we build up product, the reverse reaction becomes more likely. To ensure that we do not shift equilibrium, we try to avoid product build up. So, we want to stop reactions when we have an adequate to abundant supply of a compound.

The goal of our energy harvesting pathways, of which glycolysis is part, is the production of ATP. So ATP becomes a regulator. Phosphofructokinase has an allosteric site that is specific for ATP. When there is an abundant supply of ATP, some of the ATP binds to phosphofructokinase and changes its shape. Specifically, it closes the active site. The presence of AMP in the cell is a singal that we need to unlock our energy harvesting pathways, specifically, unlock phosphofructokinase. In this case, AMP acts as an activator to reverse the inhibitory effect of ATP. To the left is a diagram of phsophofructokinase. The area labeld "Regulatory Site" is the Allosteric site.
In lecture, we will discuss inhibition at the active site.

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Additional Resources

Enzymes at Blobs.org - This site has a good review of enzymes, including regulation, inhibition, kinetics and enzyme function at different temperatures. The images presented on the site are extremely helpful.

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Daily Challenge

Discuss the concept of enzymes using angiotensin converting enzyme (ACE) as your example. ACE is a medically important enzyme that is involved in an imporant signal system in the body that helps to regulate water balance and the kidneys. There is a group of drugs known as ACE inhibitors that affect the activity of this enzyme. Your goal is to discuss the enzyme, including its regulation and inhibition.

Note:  ACE converts the plasma protein Angiotensin I into Angiotensin II (consider this the active form).  Angiotensin II has numerous effects, including causing the kidneys to retain sodium and water, increasing blood volume, and thus increasing blood pressure.  ACE Inhibitors are used with people who have high blood pressure.

Daily Newsletter: September 24, 2013 - Redox and Coupled Reactions

Daily Newsletter

September 24, 2013

Redox and Coupled Reactions

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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.

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Daily Challenge

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, 2013 - Adenosine Triphosphate

Daily Newsletter

September 23, 2013 Adenosine Triphosphate

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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 + P_i 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).
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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.

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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

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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

Daily Newsletter: September 23, 2013 - Thermodyanmics

Daily Newsletter

September 24, 2012 Thermodynamics

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Remember that modern molecular biology is based on chemistry. 
What makes up cells?  
Chemicals. You have water, ions, proteins, carbohydrates, lipids, nucleic acids, and other compounds. Together, all of these chemicals make up what we see as the cell; but remember, the cell is greater than the sum of its chemical parts. It is the interaction of these chemical parts that makes the cell unique.
Biologists look at the chemistry of the cell from a different perspective, or lens, than chemists. In order to understand each individual chemical reaction, chemists will isolate a specific reaction, measure it, manipulate it, and report on it. Our knowledge of biochemistry and molecular pathways are all based on this reductionist perspective (reductionism - simplifying complex systems to component parts for study). Biologists take the information on these parts and attempt to visualize the whole from the individual parts. Due to this, we have to look at how the perspective of biologists differs from that of chemists when we start discussing cellular energetics and chemistry.
Today you are asked to reflect upon the topics of thermodynamics and equilibrium as they pertain to biology. These are topics originally introduced in chemistry, so you may want to go back to your chemistry books to refresh your memory. While biochemists may use these concepts unaltered from their original meaning in chemistry, most biologists look at these two concepts from a slightly different perspective.

The Laws of Theromodynamics: (the two important ones for biology are in bold).
0. If two systems are in thermal equilibrium with a third system, they must be in thermal equilibrium with each other.
(seems obvious to us know; sometimes shows up in biology).

1. Energy can neither be created nor destroyed, but can be changed from one form to another. (First Law)
• We are constantly moving energy around in living systems.
• The most obvious example of this will be the conversion of Light Energy to Chemical Energy in the production of Glucose.
• Examle: Phototrophs are organisms that can convert light energy to chemical energy.
• Example: Motile organisms (such as animals) can convert chemical energy into mechanical energy.
• Example: Luminescent organisms can convert chemical energy into light energy.
• Example: Heterotrophic organisms can convert one type of chemical energy into a different type of chemical energy. (Remember the First Law includes the conversion of Chemical Energy into a different "form" of Chemical Energy.)
2. In all energy exchanges, if no energy enters or leaves the system, the potential energy of the state will always be less than that of the initial state. (Second Law)
• This is big for biology, but not often discussed.
• You will remember this as the law of Entropy.
• Every time we experience an energy exchange, some of that energy is used, but the rest is lost due to entropy.
• Let's say you eat a muffin: Only a fraction of the chemical energy in the muffin will be harvested as useful energy by your cells. The rest is lost to entropy.
• Heat is a common disorganization of energy, i.e., entropy; but we don't want to over heat cells. (How do we get around this?)
3. All processes cease as temperature approaches absolute zero. (not generally a concern to biologists).

Later in the week, we will discuss redox reactions, but for now I want to put this concept into your heads: One of the most critical energy exchanges in biology will be the production of reducing potential.

The second law of homeostasis plays a role in homeostasis. With the second law, we know that energetically, we can never break even. Every time that we undergo a chemical reaction, we loose energy. This loss is generally going to be as heat, which is also not good for a cell (too much heat, and the cell boils). So we have be as efficient as possible knowing that we are always loosing energy. How does this play out?

• Plants take in sunlight, and make glucose.
• The sunlight is high energy.
• The glucose is going to have to have less energy because we used photons to excite electrons (an energy change).
• Animals eat plants.
• We catabolize the glucose for energy (reducing potential).
• Do we get 100% of the energy in glucose? NO.
• Animals build biomass (make proteins, lipids, etc... as needed for life).
• The energy we got from glucose is further lost when we make new biochemicals.
• We constantly need energy to keep rebuilding ourselves.
• We constantly need energy inputs to maintain homeostasis.

So why is the second law so important? To answer this question, we need to look at chemical equilibrium. You will recall this concept from chemistry, and it deals with reactions. On the right is an image of an Iron Thiocyanate reaction that was produced by the BBC. It shows the forward and reverse reactions. As you will remember, at a given set of conditions (such as a constant temperature and pressure) you can expect to see an equilibrium reached in which you will have some quantity of substrate and product. While the forward and reverse reactions will continue to occur, there will be a fairly constant quantity of substrate and product.
You may recall that one way to influence the equilibrium is to either add more substrate or remove the product. The removal of product is important for biologists, and sets up a very important condition.
In metabolic pathways, the product of one reaction becomes the substrate for the next reaction. We are constantly taking products to the next reaction. There is a constant flow, at least while an organism is alive, moving product to become the substrate of the next reaction. In biology, it is not about a single reaction, it is always about a series of reactions. This is a critical point! A chemist may look at a single reaction, but a biologist must look at the overall set of reactions if they are to understand the organism.
So, an organism is constantly alterating the equalibirum of a given reaction by taking the product and using it as a substrate in another reaction. We will also see that organisms are constantly acquiring energy and "building block". As such, organisms never acheive the equilibrium a chemist would see in a test tube, and we constantly acquire energy to avoid entropy (consider starvation where you have no new inputs of energy, what happens to the system?).

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Daily Challenge

Today you are tasked with describing in your own words how the laws of thermodynamics and equilibrium play out in living systems. Use the Newsletter and our class discussions as the start point, but generate your own analogies and examples. 

Daily Newsletter: September 19, 2013 - Membranes: Fluid and Dynamic

Daily Newsletter

September 19, 2013 Membranes: Fluid and Dynamic

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Today's news letter is different in that it holds three background articles from the 2004 Horizon Symposium.  This Nature sponsored symposium dealt with cellular membranes.  The three article are a good introduction to scientific communication, and will help you better appreciate the cell membrane.

• Mind the membrane 
• The need for fat  
• Membranes and the diseases within  

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Daily Challenge: The Fluid Mosaic Model 

 Using the three articles above as references, discuss the fluid and dynamic nature of the membrane in terms of the fluid mosaic model.  For example, address why it is critical that the membrane is composed of "proteins floating in a sea of lipids," and why is it important that the membrane is dynamic (ever changing?) ?

Daily Newsletter: September 18, 2013 - Active Transport

Daily Newsletter

September 18, 2013 Active Transport

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In active transport, cells are moving substances across the membrane, but against the chemical concentration gradient. Chemicals are move from areas of low concentration to areas of higher concentration. To move against a concentration gradient requires energy to overcome the inherent Brownian motion of the molecule. This always requires protein (enzyme) pumps. The word pump implies an active process that moves against a natural gradient or flow. (consider: passive transport proteins are called channels, pores or carriers, while active transport proteins are called pumps)
The most commonly discussed pumps will be the ion pumps. There are two pumps that all biology students must become familiar with as they are critically important and are discussed in many different biology courses.

• Sodium-Potassium ATPase (also known as the Na⁺/K⁺ pump).
  • This helps to establish and maintain the Sodium and Potassium gradients of a cell.
  • Sodium should be at high concentrations outside of the cell (extracellular)
  • Potassium should be a high concentrations inside of the cell (intracellular)
  • This combined gradient helps to establish the Resting Membrane Potential of many cells (an electrical charge across the membrane).
• Proton Pumps
  • This pump system help to establish and maintain a proton (hydrogen ion) gradient.
  • In Eukaryotes, this will be found along the inner mitochondrial membrane.
  • In Prokaryotes, this will be found along the cell membrane.
  • This is a critical electrochemical gradient for cellular energy.

With both of these pump systems, we are creating electrochemical gradients, and both represent potential energy.

All active transport systems require energy to work. Primary active transport will utilize ATP, while secondary active transport will utilize either reducing power (redox reactions) or an established electrochemical gradient.

Primary Active Transport: The addition of the phosphate causes a conformational change in the proteins structure. Remember, you are adding a -2 charge to a specific location on the protein; this will change the electrical profile of the protein, causing the proteins folding pattern to change. Note that both Na⁺ and K⁺ are moving against their electrochemical gradients.
Secondary Active Transport: As with active transport, there will be a conformational change in the protein (aka, the folding pattern, and hence shape, will change). The cause of this shape alteration will either result from a redox reaction or an established electrochemical gradient. In the image below, we see the entry of one ion (sodium) causing the expulsion of a second ion (amino acid) in an antiport system. How do you know that sodium is the "motive force"? Look at the Na⁺ concentration gradient; sodium is moving down the electrochemical gradient.

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Important Memes

When an ion moves down it's electrochemical gradient, across a membrane, work (kinetic energy) is done. The electrochemical gradient is a seperation of charged particles (ions) on either side of the membrane. When we speak of a Sodium (Na⁺) gradient, we are actually referring to an electrochemical gradient. Movement of ions down an electro chemical gradient is akin to hooking up a battery in a circuit, you get kinetic genery (work is done). Until then, the electrochemical gradient acts a potential energy.
When you add something to a protein, the protein changes shape. 
Whether it is the substrate of an enzyme, an ion, or a phosphate, as things are added, the protein's shape changes.

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Daily Challenge: Concentration and Electrochemical Gradients

Today, reflect on the idea of concentration and electrochemical gradients.  In living system, we do not see the typical end point of diffusion in which you get equal concentrations on either side of the membrane.  Instead, we are constantly adjusting concentrations by using membrane proteins (Such as the Na⁺/K⁺ pump above).  Why is it necessary for cells to do this?  Why is it that disruption of the membrane (and therefore the possibility of concentration equilibrium) means the death of a cell?

This article from Scitable may help:   Why Are Cells Powered by Proton Gradients?

September 17, 2013 The basis of the cell membrane

Daily Newsletter

September 17, 2013 The Basis of the Cell Membrane.

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Nature of the Cell

The cell theory describes the importance of the cells to biologists. How important is it? Well the first part of the cell theory answers that: All known living things are made up of one or more cells. But what ultimately is a cell? What takes place in one? Why are they so important?

It will take a few discussions to get to all these questions, but there is a starting point. As a basic description, the cell is a self-managing, self-contained chemical factory. Cells take in materials, use these for energy and building blocks, and then produce materials to keep the cell healthy, harvest nutrients, eliminates waste, and produces products. The cell has many components to accomplish these tasks: DNA for management, chemical signals to send messages, enzymes for chemical activity, etc....

Let's look at the analogy of a chemical factory: Inside a factory, there are going to be different processing places for different chemicals. There are going to be pathways of pipes going between vats and other structures. Taking a further look back, there is a building, with trucks coming and going.

Inside of the cell, chemical reactions will be taking place. Outside the cell, chemical reactions are taking place. Are they the same chemical reactions? One of the foundations of cells is that the inside of a cell is a spatial area with a defined concentration of a variety of chemicals that is distinct and different from the chemical concentrations found outside of the cell.

So, there is an inside and outside of a cell, and they are different.

You will hear me say again and again that the cell membrane is the defining structure of a cell. Why? Because it establishes the boundary. When you have a cell membrane, you can have an inside as opposed to an outside of a cell. If you loose that membrane, you start moving to a full equilibrium between the inside and outside of the cell. If you loose the membrane, or it gets holes, the cell dies. The cell dies when you cease to have an inside vs. an outside.

The fastest way to kill a cell is to poke holes in it.

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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.

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Fundamental Structure of the Cellular Membrane

The basic structure of the cellular membrane is composed of Phospholipids. The amphipathic nature of phospholipids means that they will naturally associate with one another. Specifically, the hydrophobic tails want to be with other hydrophobic compounds, and exclude polar compounds (Hydrophobic Exclusion-This is a good concise discussion of the topic by Stephen T. Abedon, Ph.D. at Ohio State). Below is a brief movie that shows what happens when phospholipids in water begin to interact.

Phospholipid Movie: Bilayer formation through molecular self-assembly

The resulting phospholipid bilayer is polar (hydrophilic) on the outside, while the middle is non-polar (hydrophobic). The sides interact with water, but the middle excludes polar substances. This creates a selectively permeable barrier, and is the basis of the membranes function. Changing phospholipids and adding sterols (like cholesterol) will change the integrity and stability of this basic membrane structure.
Selective permeability means that only certain classes of chemical can make it through the phsopholipid bilayer. For other chemicals, we need to provide a protein to serve as a pore, channel or transporter.
In general, there are two ways that a chemical can be moved across the membrane: Down the chemical's concentration gradient (diffusion), or Against the chemical's concentration gradient. When a chemical moves down it's concentration gradient, we do not need to add energy to the process. The concentration gradient and the kinetic energy of the molecule (Brownian Motion) provided the needed energy. We call this form of movement Passive Transport.

There are three basic forms of passive transport through the membrane:

1. Diffusion - Using the inherent Brownian motion of molecules, chemicals move from points of high concentration to points of low concentration.

• Every chemical has a unique concentration gradient.
• The concentration gradient of one molecule will not interfer with the concentration gradient of another molecule.
• What interfers is the ability to move across the cellular membrane (phospholipid bilayer).
• Molecules with high polarity, ions, and large molecules are excluded by the phospholipid tails, and thus can not cross.
• Water*, CO₂, O₂, and nonpolar compounds (lipids) can cross though diffusion.
• Water moves through very slowly; water moves across more readily due to porins (protein pores) possessed by cells to make sure water and small polar compounds can cross readily.

Osmosis - The movement of water across a selectively permeable membrane.

• Water goes to where the party is, meaning it will move to a compartment that has a higher solute concentration.
• Since we are talking about two fluid compartments on either side of a membrane, we are ultimately talking about relative solute concentrations.
• REMEMBER: you are looking at two fluid compartments, so when we are looking at osmosis, we are discussing the movement of water across a selectively permeable membrane from one fluid compartment to a second fluid compartment.
• In biology we always use the inside of the cell as our reference, so:
  • An isotonic fluid has the same solute concentration as the inside of the cell.
  • A hypertonic solution has a solute concentration that is higher than the solute concentration inside the cell.
  • A hypotonic solution has a solute concentration that is lower than the solute concentration inside the cell.

• Remember that we are looking at solute concentrations, not the concentration of a single chemical.
  • Diffusion gradients are specific for each chemical.
  • Osmosis is determined by total solute concentration on each side of a selectively permeable membrane.

Facilitated Diffusion - Ultimately, this is diffusion, but the cell has had to provide a passage for the chemical. So, this only applies to chemicals that can not normally pass through the phospholipid bilayer.

• The cell provides a protein channel or pore for the chemical to pass through.
• Each channel or pore is specific to a single chemical or set of chemicals.
• Since this is an protein (enzyme) mediated action, the ability to transport follows standard enzyme kinetics.
• The core idea about facilitated diffusion following enzyme kinetics is that diffusion becomes limited by the number of channels or pore available.
• Note: You will generally build up a large concentration of a compound on one side of the membrane. We will refer to this as a steep gradient (high on one side, low on the other).
• The facilitator proteins will allow molecules from the HIGH side to move toward the LOW side; hence the use of the word diffusion.
• Rarely will the channel or pore (facilitator) allow transport in the reverse direction for the same ion.

In each case, the cell does not need to expend energy to move the chemical across the membrane. The motive force is built in to the chemical gradients. Remember, the cell membrane is going to provide an internal vs. an external space (compartment). Each side of the membrane will have a unique chemical profile. As such, there will be concentration gradients across the membrane.

Osmosis Movie

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Important Memes

Occassionly, there will be a phrase that I want you to remember to help you as you move through biology.

1. Water goes to where the party is! (This is a great way to remember in what direction water will flow across a selectively permeable membrane in response to changes in osmolarity, aka, solute concentration).
2. The phospholipid bilayer establishes the structure of the membranes, but the proteins provide the function. (The proteins embedded in the membrane will determine the functional capabilities of the membrane).
3. When something is added to a protein, the protein changes shape.

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Daily Challenge: Nature of the Cell

A critical concept for biologists and biochemists is that we never see a reaction or molecular movement that results in a final equilibrium, as you would see in a chemistry lab.  The cell is constantly moving substances across the membrane, actively maintaining concentration gradients, and quickly using products of chemical reactions.  Water is one of the few compounds we do not use/regulate in this way.  Instead, water is regulated by solute concentrations.  When solutes are moved from one fluid compartment to another, water follows.  The cholera toxin offers a great example of how water moves when we move solutes.

The cholera toxin forces intestinal endothelia cells (cells that line the intestine) to purge Cl^- into the lumen of the intestine (hollow tube). Water follows.  The end result, diarrhea. 


Your goal today is to reflect on two different concepts:  1)  the formation of fluid compartments (at minimum the inside and outside of a cell) as critical to the life of the cell, and 2) how the cell maintains a dynamic equilibrium among all the chemicals it uses.

Daily Newsletter - September 12, 2013 - Translation

Daily Newsletter

September 12, 2013 - Translation

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The core concept of translation is the connecting of a codon to an amino acid. As we saw yesterday, this 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 (holozyme) 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 neccesary 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? It's anticodon is complimentary to the a 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 comples. 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:

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 aminom 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 to short and many 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 seperate the ribosomal subunits. KEY CONCEPT: The stop codon signals the end of the coded message.

Additional Information

The following is just a little additional information about transcription.  This "special topic" is not going to be used for tests/exams, but we will see this material again before the end of the semester. Once completed, proteins can be further modifed 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 Golig 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 less extensive posttranscriptional modification in prokaryotes, but they have numerous proteins that do require modification].

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Daily Challenge

Proteins provide the cell with various functions, from channels that allow the passage of ions to enzymes that govern catabolic processes.  In translation, a sequence of nucleic acids is translated into a sequence of amino acids.  The ribosome is central to the process of translation, and though the size and complexity vary between domains (archaea, bacteria, eukarya), it works basically the same.

Reflect on the universality of the genetic code and the ubiquitous ribosome.  SNPs were mentioned yesterday; what would happen if you altered the sequence of amino acids in Ribosomal proteins?  What would happen if you altered the sequence of nucleotides in Ribosomal RNA (rRNA)?  Really consider how vital the process of translation is to the life of a cell.  The only time we slow down making proteins when during cellular division (even then we don't stop making proteins).

Write a description of the process of translation, and include your insights into this vital metabolic activity.
Link to Forum

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Special 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 resarch, and make sure that you provide a description of both the geneome and the proteome of an organism.
Link to Forum

Daily Newsletter - September 11, 2013 - Codons, Anticodons and Amino Acids

Daily Newsletter

September 11, 2013

Codons, Anticodons & Amino Acids

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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?

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Daily Challenge

On September 5th, the newsletter 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 10, 2013 - Transcription

Daily Newsletter

September 10, 2013 Transcription

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Transcription is the genetic process where a single strand of DNA acts as a template for the construction of a complementary RNA strand. Generally when talking about transcription, we will be talking about the formation of messenger RNA (mRNA), which carries the code for one gene to a ribosome where it is translated into a protein.
DNA holds the "permanent" copy of the genes needed to make a functional organism (nothing is really permenant). Think of DNA as a locked safe where you hold all your company's blueprints, patents and documented procedures. You don't want to loose these, or risk that they might be changed. You only bring them out to make copies of them, then they go back to the safe. This is what happens with your DNA. You keep it tightly locked up (in a double-helix that is coiled around histones, and then possibly supercoiled), and open it up only when you NEED to make a copy. Notice how NEED is highlighted? Do you think it might be an important concept?
In eukaryotic DNA every gene starts with a promoter. This is a sight of ~8 nucleotides visible in the major groove of DNA. The transcription complex recognizes this sequence as a "START" indicator. The main core of the transcription complex will be RNA polymerase. This enzyme works to build a strand of RNA complementary to DNA. The name polymerase indicates that it is involved with dehydration synthesis polymerazation reactions (taking one nucleotide, and adding it to a growing chain of nucleotides). Like DNA polymerase, RNA polymerase builds in the 5' to 3', and builds phosphodiester linkages between nucleotides.
But RNA polymerase can not act alone. In eukaryotic systems, initiation factors are needed to recognize the promoter region, and then to correctly align the RNA polymerase. To the left is a great picture showing the initiation complex and the RNA polymerase II holozyme (RNA polymerase II with all associated protein structures). You are not responsible for knowing all of the factors needed to initiate eukaryotic transcription, but you do need to start understanding the concept that it takes multiple factors to identify a promoter and start RNA polymerase. What do you think you need in order to recognize a specific sequence of nuclotides?
As you can see, TATA Binding Protein (TBP) is the first structure to attach to DNA. It recognizes the TATA sequence in the major groove of the DNA double helix. It then forces the the DNA to bend, and acts as a signal to other enzymes directing interactions with DNA. A cascade of reactions occur to then produce the Preinitiation Complex, which ensures that the transcription complex is positioned correctly over the Transcription Start Site, and begins the unwinding (sometimes referred to as denaturation) of the double helix. The Transcription Complex then begins to read the template strand of DNA, and makes an RNA copy (Elongation). [NOTE: Bacteria use proteins known as sigma factors to help find promoter regions and initiate transcription. There are different sigma factors linked to different environmental and physiological states, such as the Heat Shock Sigma factor which alter's the bacteria's ability to deal with higher temperatures)]
Elongation works due to base complementarity. Ribonucleotide triphophates are brought into the transcription complex, and are added to the free 3' end of the growing RNA strand. During the elongation phase, the RNA polymerase continues to add nucleotides to the growing RNA strand.
At some point, the RNA polymerase comes to a termination sequence. We are not going to spend a lot of time on termination (you are not held responsible for the various models). There are a couple different models of eukaryotic transcription termination. The main feature is that there is a signal sequence of deoxyribonucleotides in DNA that signals the end of transcription. Once this signal sequence is found, RNA polymerase is removed and the new transcript (new RNA molecule) is released.
mRNA processing: Once transcription is complete, in eukaryotes, the RNA needs to be processed. The following is a quick reference for mRNA processing:

• 5' capping: To protect the mRNA from ribonucleases (RNA degrading enzymes) that attack the 5' end, 7-methylguanosine is added to the 5' end. Usually, the 5' ribonucleotide is replace by this compound. Additionally, methyl groups can be added to the sugar-phosphate backbone to further protect the mRNA.
• Polyadenylation: In maturing RNA to mRNA, a poly-A tail is added (usually after cleaving off a small section of the 3' end). This process adds ~250 adenyls to the 3'end of the molecule. This is needed to stabilize the molecule and facilitate export through the nuclear pores. As mRNA is translated, the poly-A tail gets shorter. When short enough, the mRNA is degraded. Thus, the polyadenylation (poly-A tail) is responsible for setting a time limit to the mRNA. 
• Splicing: The RNA is composed of both coding (exon) and non-coding (intron) regions. To mature into mRNA, the introns have to be removed, and the remaining exon spliced together. This job is the responsibility of the splicosomes.

• The above image is a quick reference to the effects of splicing.

 

• The above image is a quick reference to the effects of the splicosome.

Once RNA has been processed (matured), it is ready to be used in translation (protein synthesis). NOTE: Bacterial RNA does not undergo processing. The bacterial RNA transcript is immediately translated.

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Daily Challenge

Transcription In your own words, discuss the process of transcription, and the formation (maturation) of mRNA. Remember that we have focused on eukaryotic transcription. Briefly, how does prokaryotic (specifically bacterial) transcription differ from eukaryotic transcription?
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