Biochemistry Final Flashcards
Why is glycogen important for the body during exercise?
What does “hitting the wall” mean?
Your muscles store up glycogen and use it during exercise.
The amount of glycogen in your muscles, and your body weight, determine how far you can run, easily.
“Hitting the Wall” is a term for how one feels when the glycogen runs out. The length of a Marathon (26.2 miles) more or less guarantees that everyone should “hit the wall” before finishing.
What is the purpose of a cascade? Name some parts of the pathway.
Each step turns on or enables the next step.
The start of this pathway is the 7TM receptor, the beta adrenergic receptor, followed by G proteins and Adenylate Cyclase.
What does this picture represent? What is the yellow molecule in the middle?
This represents the very large molecule, Glycogen. The yellow ball at the center is a protein, Glycogenin.
But the reducing end is protected by attachment to a Serine of Glycogenin.
Glycogen is a _________ of glucose
Polymer
About 90% of the linkages are alpha 1-4, but about 10% of the links are alpha 1-6, those are the branch points.
Glycogen granules are so large that they can be seen with a __________ __________.
Light microscope
Why is Gluocse 6-phosphate important?
Responsible for glycolysis and glucogeneosis
Glycogen>Glucose>G6P
What is the main enzyme that breaks down glycogen and what can it only cleave? What is the advantage of using it?
Glycogen Phosphorylase (or Glycogen Phosphorylase a).
Phosphorolysis cleaves a molecule using phosphate. The advantage of doing this is that you immediately get a sugar phosphate without having to use an ATP.
Glycogen Phosphorylase can only cleave alpha 1-4 linkages to sugars 4 or more residues away from a branch point.
Why doesn’t glycogen phosphorylase compete well with hydrolysis?
Phosphorolysis would not compete well with Hydrolysis and that is why the deep active site is desirable, so water can be excluded.
1) the active site is DEEP and 2) the glycogen binding site is “around the corner” from the active site.
The phosphate of PLP helps to position the phosphate that will be used in Phosphorolysis
What is this structure and why is it important?
This is the structure of PLP bound as a prosthetic group to Lysine of the Phosphorylase enzyme.
Why are the intermediates important in this reaction?
The branch point glucoses can be removed by a hydrolase.
Alpha 1-6 Glucosidase is also known as “Debranching Enzyme”
It releases free glucose instead of Glucose – 1 – P
Thus about 10% of the glucose from breakdown of normal glycogen is the free sugar while about 90% of the glucose is found as G-1-P or G-6-P.
- _____ ____ takes off all the red glucose residues, then it must stop (too close to branch point).
- THEN blue residues are moved from the branch to the end of the long main chain by a __________enzyme.
- THEN green residue, the branch point glucose, is removed by Debranching Enzyme ___ __ __ _____.
- Now the resulting long chain of ________,__,__ linked glucose can be broken down by Glycogen Phosphorylase
- Glycogen Phosphorylase
- Transferase
- alpha 1,6 Glucosidase
- alpha 1,4
___________ reversibly transfers a phosphate so that Glu-1-P = Glu-6-P and the other direction
Phosphoglucomutase
Whats the difference between an R and T state?
Whats the difference between Phophorylase a and phosphorylase b?
Phosphorylase _______ covalently activates Glycogen Phosphorylase. (in the liver)
R- Relaxed (Activated)
T- Tense (Inactivated)
Phosphorylase a- Always active, has phosphoserine residues, mostly in R state
un-phosphorylated “b” form- Low energy charge can allosterically activate the “b” form, mostly in T-state (muscles)
Kinase
What is happening in this picture?
Phosphorylation of the 2 serines causes a conformational shift. The blue and green hairpins at the BOTTOM are poking into the little red holes.
That represents a loop of protein hindering the active site, therefore = inactive.
The blue and green hairpins at the TOP are pulled away from the little red holes – thus the active sites are free and open in the R state and that means = active.
What is happening here with phosphorylase a?
The phosphorylated “a” form is nearly always active – but high levels of Glucose can allosterically inactivate the “a” form.
What is happening here with phosphorylase b?
This shows the allosteric (non covalent) change from inactive “b” form (on the left) to active “b” form on the right.
As mentioned before low energy charge = higher AMP causes a shift from T to R with no phosphorylation, producing active “b” form
Presence of high energy charge (ATP) or high levels of the product of Glycogen Breakdown (Glu-6-P) cause a shift back to the left inactivating the “b” form. Going from inactive to active.
What is the point of this image?
____________ __________ is the enzyme that activates Glycogen Phosphorylase in the Glycogen Breakdown Cascade.
Phosphorylase KINASE
The point of this image is that 1) phosphorylation produces partial activation, 2) the presence of calcium produces partial activation, and 3) BOTH TOGETHER produce full activation
(Calcium is especially important in muscle contraction – so part of what we see in exercising muscle is Phosphorylase “b” which is active because of low energy charge)
Both ______ and ________ trigger the process of Glycogen breakdown in the Liver, and then the liver exports glucose into the blood.
Glucagon and Epinephrine
What is happening during the glycogen breakdown cascade?
Adenylate Cyclase is activated first
Draw the structure of Epinephrine
What is Glucagen?
A small protein or large oligopeptide and that it triggers Glycogen breakdown in the liver.
Why is UDP Glucose important?
It is the starting material for Glycogen Synthesis.
Whenever you want to do something interesting with Glucose (like polymerizing it into Glycogen) you need a UDP handle
Draw out how UDP Glucose is made
FIRST you take G6P and convert to G1P with the enzyme Phosphoglucomutase.
Then you take UTP and the enzyme Uridylate Transferase. “Uridylate” is how you pronounce UMP. The UMP (blue in the slide) moves from UTP onto G1P leaving a pyrophosphate behind (black).
How is UDP AND Glycogen made from UDP-Glucose?
UDP Glucose acts as a substrate for Glycogen Synthase, adding to the growing chain by making an alpha 1,4 bond.
If you ONLY use Glycogen Synthase, then you don’t get Glycogen – it would be a linear molecule.
What is happening in this picture?
Glycogen Synthase has added several glucose residues (blue) to the initial “core” chain. And what has been made is a linear starch molecule.
The remodeling enzyme is called Branching Enzyme – it breaks the alpha 1-4 bond and moves the “blue” segment inward to form an alpha 1-6 bond, a branch point.
Here you see seven residues being moved to a spot more than 4 residues from the end of the chain and more than 4 residues from the nearest branch.
What does the yellow “G” reresent?
This is a schematic of a small Glycogen molecule showing G for Glycogenin – the protein at the center of the Glycogen, attached to the reducing end of the molecule.
How do you reverse what the Cascade has done?
The serine phosphates are broken down by Protein Phosphatase I. The arrows coming up from the bottom represent the relaxation of the Cascade.
The arrows coming down from the top represent the activation of the Cascade by Epinephrine or Glucagon, but PPI, Protein Phosphatase 1, counteracts those.
What are the Glycogen storage diseases I, III, and IV and their defective enzymes?
Von Gierke’s Disease, Cori’s Disease, and Andersen’s Disease.
Glucose-6 Phosphate, Amylo 1-6 Glucosidase, Branching enzymes 1,4 and 1,6
In what animal were metabolic and behavioral differences found in?
Mice
What is the difference between a fat and an oil?
A fat is generally a triglyceride which is solid at room temperature.
An oil is a triglyceride which is liquid at room temperature.
Fats tend to be composed of TG of saturated fatty acids = no double bonds.
Oils tend to be composed of TG of unsaturated/polyunsaturated fatty acids = with double bonds.
What are adipocytes?
Because oil and water don’t mix – adipocytes, or cells from fatty adipose tissue, have a fat globule surrounded by a “skin” with the rest of the cell, cytoplasm, nucleus, mitochondria
The structures and processes of fatty acid beta oxidation (on left) are very similar to those of ________ ______ ________.
Fatty acid synthesis
Why do birds store fat?
Migratory birds add a layer of fat before they start their long flights of 25,000 miles. This is an American Golden Plover.
What makes lipase enzymes important?
Fats are broken down by Lipase enzymes which are under hormonal control. The adipose tissue is dispersed around the body but acts as an organ because of the hormonal control.
What happens during absorption from the intestine?
What are Chylomicrons?
Fats are broken down and then re-built
Chylomicrons are essentially large fat globules surrounded by a “skin” of protein. Protein weighs more than fatty acid
Again, what does Lipase do?
Why are free fatty acids bad for you?
Lipase breaks down TG into glycerol plus fatty acids
Free fatty acids are “bad for you” because they can act as uncoupling ionophores in mitochondria. So it is important to get something on the carboxyl groups promptly and not allow too many free fatty acids to build up in the cell.
Lipase is under “_________ _______” and that includes _____ and _____ _____ __.
Hormonal control, cAMP, Protein Kinase A
When triglycerides are hydrolyzed the glycerol and fatty acids go in ______ and _____.
Liver Cell and Muscle Tissue
How can a thioester bond between a fatty acid and CoA form?
You want a leaving group, a way to put some energy into the process. And ATP “donates” an Adenylate (AMP) leaving group.
When the CoA goes on, the AMP comes off and it all works smoothly.
The enzyme is called Acyl CoA Synthetase, or Fatty Acid Thiokinase.
What is Acyl Adenylate and what can’t it do?
Acyl AMP
Can’t cross mitochondrial membrane so we need the carrier carnitine
How does Acyl coA get into the mitocondrial matrix?
Get in with The enzyme is Carnitine Acyl Transferase I. Attaching a fatty acid to Carnitine allows it to cross the mitochondrial membrane.
Acyl CoA is first formed in the cytoplasm – but if we want to “burn” our fatty acids they have to get inside the mitochondrial matrix.
If a fatty acid enters the matrix, it will be oxidized to make more ATP.
Thus when fatty acids are being synthesized in the cytoplasm, say for membrane lipids, the reaction shown here has to be inhibited.
What happens inside the matrix with regard to Carnitine Acyl Transferase II?
Inside the matrix, Carnitine Acyl Transferase II takes the fatty acid and puts it back onto Coenzyme A.
Now we are ready for oxidation – Fatty Acid Beta Oxidation
How was Fatty Acid Beta Oxidation named?
It was noticed that the “beta” carbon received a hydroxyl and then a keto group, the beta carbon was oxidized.
The processes of beta oxidation are VERY SIMILAR to the steps of the Citric Acid Cycle from Succinate to OAA.
How does Acyl CoA DH resemble Succinate DH?
One of the ways in which Acyl CoA DH resembles Succinate DH is that it does exactly the same thing with the electrons taken from substrate.
Write out the steps from Succinate to OAA (Citric Acid Cycle) side by side.
Similar to CAC and same cofactors
(Slide 50)
What do each of the enzymes that are listed do?
In the CAC, you _____ an Acetyl CoA.
But in the Fatty Acid Beta Oxidation sequence you ________ an Acetyl CoA. Each cycle removes 2 more carbons.
ADD
SUBTRACT
How do we fix delta-odd double bonds? (to become even)
With a pre-existing double bond there is one less _______ produced so the ATP yield is diminished by _____ ATP
Isomerase
FADH2
1.5 ATP
(Look up in phone delata and even and odd strutures he drew)
What is fatty acid C-17?
An odd fatty acid chain. Mainly comes from seafood.
Beta oxidation of odd chain fatty acids goes down to 3 so you get Propionyl CoA
Draw the pathyway from Propional coA to Succinyl coA
How do you fix an L Isomer?
If you could JUST add a carboxyl group to Carbon 3 of Propionyl CoA, you would have Succinyl CoA and the problem is solved.
BUT the carboxyl goes on at Carbon 2 (because of electron sinks etc.) instead, giving you Methyl Malonyl CoA.
And if only you had gotten the L isomer of MeMalCoA you could “fix” it with B-12, but the reaction gives you the D isomer instead.
So you need a “racemase” enzyme to convert D to L.
Finally “cobalamin” or vitamin B-12 “magically” solves the problem the carboxyl-CoA group onto the methyl group.
What does vitimin B12 resemble?
That its a heme group and a 1 carbon carrier
Draw out Ketone Body Synthesis and describe what it is.
Why can’t we just hydrolyze off the CoA from Acetoacetyl CoA?
- Just as the liver sends out Glucose to nourish the other organs, it also sends out Ketone Bodies.
The main Ketone Body is Acetoacetate, but it can be reduced to Hydroxybutyrate or decarboxylated to Acetone.
- Because this would be another “dangerous” enzyme.
You can’t go around hydrolyzing CoA derivatives, they are too expensive to make in the first place.
So first we ADD another Acetyl CoA to make HMG CoA, 3-hydroxy-3-methyl-glutaryl CoA. HMG CoA is the starting point for terpene synthesis.
And HMG CoA HMG CoA Synthase (enzyme “2”) is the target for Statin drugs that lower cholesterol.
Adding one Acetyl CoA and subtracting another Acetyl CoA leaves us with Acetoacetate.
Why is using Succinyl CoA as the CoA donor smart?
Draw how Acetoacetate produces 2 Acetyl coA?
It only takes one GTP hydrolysis to put the CoA back on to Succinate.
Thiolase or Ketothiolase is also the last enzyme in Fatty Acid Beta Oxidation.
Draw out how 3 hydroxybutyrate is oxidized to Acetoacetate
Name 3 examples of how ketosis is triggered
1) Starvation – glycogen and other carbohydrate sources are quickly used up so that the body has to use fats for energy.
2) Diabetes, as shown above. If muscles can’t absorb glucose then they must use fats and ketone bodies.
3) Fad Diets like the Atkins Diet. By severely restricting carb intake, these diets force the body into Ketosis.
Ketosis makes your breath smell like acetone, and it can damage your kidneys (acid accumulates in kidneys).
One can die from severe ketosis.
ACP is functionally the same as CoA but how are they different?
ACP “anchors” it to the enzyme complex in the Cytoplasm
Describe what happens in the Citrate Lyase Cycle during fatty acid synthesis regarding Acetyl CoA in the mitocondria and the cytoplasm?
During fatty acid synthesis Acetyl CoA has to be moved from the mito matrix to the cytoplasm.
It is done by exporting Citrate, which is then broken down to give Acetyl CoA and OAA.
The cycle also produces NADPH which is needed for fatty acid synthesis.
How does Phytanic acid enter a person’s diet? What membrane is it a part of?
It enters the diet with green plants because it’s part of chlorophyll. Phytanic acid is also a prominent part of the membranes of ARCHAEA
Why do we say that this reaction uses up 2 ~P when as you can see only one ATP is present?
ATP to ADP is only 1 ~P, one high energy phosphate bond. But when you go from ATP to AMP that is 2 ~P because it takes 2 kinase steps using ATP to get back from one phosphate to three phosphates.
How did 14th century German’s diagnose diseases? What were diabetics called?
An experienced doctor could learn a lot from the color, smell, and taste of a patient’s urine. Diabetics were sometimes called “sweet peas.”
*On Final
Draw out a simple version of the Urea Cycle with its 4 intermediates
What are the 9 Essential amino acids human being can’t make? Which ones are Branchy, Aromatic, and weird? (PVT MATT HILL)
How can someone grow a tumor?
3 Branchy- VAL, LEU, ILE (“Bratty Valerie, Leucille, and ilani”)
3 Aromatic- TRP, PHE, HIS (“Amazing Trips, Peaks, and Hikes”)
3 Weird- THR, LYS, MET (“Welcome Thirsty Lifeless Men”)
Positive nirtogen balance and babies can’t make argenine
Proteins in general are not absorbed into the body as proteins, but are broken down into ______ ______and ______ ______.
Amino Acids and Small Peptides
What is Ubiquitin? (Don’t need to know how to draw)
Ubiquitin is a small protein which is used to mark “old” proteins for destruction. It attaches to the target protein by binding to a Lysine side chain.
How is a proteosome formed?
Several ubiquitins are added and promotes folding
The proteasome has rings of ___ subunits with a cavity in the middle.
7 (fold-barrel)
How do proteins inside the Proteosomes UNfold?
Proteins are unfolded inside the proteasome and then cut up into peptides. The Ubiquitin molecules are released and re-used.
Which proteosome is closer to us; Archaeal or Bacteria?
Achaea is closer to us
What is Transemination? What plays a key role in amino acid metabolism?
Where an amino acid plus keto acid becomes keto acid plus amino acid.
The nitrogen is “swapped” from one molecule to another.
This is how most amino acids get rid of their nitrogen – often giving it to aKG
Alpha-Keto-Glutarate plays a very important role in amino acid metabolism.
WHY is glutamate so important in the metabolism of other amino acids?
In the following picture, there are the metabolites found in E. coli growing on a diet of glucose. Look at how much Glutamate there is! Far more than any other metabolite.
What is the enzyme Glutamate DH responsible for?
It controls the rate of amino acid catabolism. There are NAD+ versions and NADP+ versions and it is reversible.
Why is the ammonia generated during amino acid catabolism dangerous? How can you make it not dangerous?
The ammonia generated during amino acid catabolism is potentially toxic, so it is made into Urea which is safer.
(My notes: Cats died without arginine in their diet because they need meat)
________ ________ is an extremely important cofactor in the catabolism of amino acids.
Draw it out.
Pyridoxal Phosphate
(When he draws it he mainly draws the six membered pyridine ring and the aldehyde on top)
How does Aldamine form into PMP?
What are the 3 possible outcomes when an amino acid binds to PLP?
Either an R, H, or COO- attaches
Because pyridine is aromatic there is one big ____ cloud which makes the molecule mostly ________.
Pi
Planar
How does serine become pyruvate? Draw the pathway
How does Alanine work in the liver and muscle? Draw the pathway.
Draw the Urea cycle and where different things take place
This schematic of the Urea Cycle shows that there is a compartment change, most of it takes place in the Cytoplasm but there is a step in the Mitochondrial Matrix.
*Remember that there are only 4 steps in the Cycle itself.
What does this picture tell us?
Ammonia is toxic if it builds up in the blood. Urea doesn’t have the same acid/base behavior and is safer.
Draw out the specifics of the urea cycle (In the cytoplasm)
Draw out the urea bicyle.
How can Fumarate be made back into Aspartate?
Aspartate enters the Urea Cycle and Fumarate leaves the Urea Cycle.
Fumarate enters the Mitochondrial Matrix and goes to Malate and OAA. Then the OAA can be transaminated to give Aspartate.
Both of the nitrogens of Urea can be shown to originate in the Mitochondrial Ammonia Pool. One comes from Carbamoyl Phosphate.
The other comes from this transamination which can get the nitrogen from Glutamate.
The PROCESS of “__________” an amino group from Aspartate to produce Fumarate is not unique to the Urea Cycle or to Argininosuccinate biosynthesis
Donating
Draw out the reaction from Proline to Glutamate AND Argenine to Glutamate
What is the common intermediate?
Glutamate semialdehyde is an important intermediate for Proline and Ornithine catabolism
Draw out the pathway from Propionyl CoA to Succinyl CoA for fat metabolism
How do you go from Phenylalanine to Tyrosine to make Epinepherine?
NADPH—–>NAD+ included as well
What is the ENTIRE process from Phenylalanine to Epinepherine?
Name these intermediates from the Urea cycle and put them in order
(May need to go back and look what the answers are)
What is the simple pathway for a-Ketoglutarate in protein synthesis?
What is the simple pathway for 3-phosphoglycerate for protein synthesis?
How do you go from Glutamate to Proline? Show the steps.
Draw the entire structure (3 parts) of THF
Why is Folic acid important?
Pteridine, p-aminobenzoate, and Glutamate
A very important vitamin. PABA = para amino benzoate is an important ingredient of sun screen. It blocks UV light in the range that causes burns.
Folic acid is found in green leaves (folic = foliage) and red meat. If you eat your spinach raw, you get a lot more chained Glutamates than if you eat it cooked to a pulp.
How do you go from Serine to Glycine in purine synthesis?
THF DERIVATIVES
What does Methyl get oxidized to?
What does Methylene get oxidized to?
What does Formyl Formimino Methenyl get oxidized to?
- Most reduced= Methanol (-CH3)
- Intermediate= Formaldehyde (-CH2)
- Most Oxidized= Formic acid (-CHO, -CHNH, CH=)
The nitrogen in the six membered ring (from Pteridine) is ____, and the nitrogen from PABA is ____.
N5 and N10
Draw out: Folic acid, N5 Methyl, N5N10 Methylene, N5N10 Methanyl, N5 Formyl, and N10 Formyl
ATP must lose ALL _____ PHOSPHATES when it reacts with Methionine to form SAM. That is because it binds to Methionine at the 5’ carbon.
Show the synthesis breakdown of Methionine
What is the first amino acid in nearly every protein?
What do you do to the methionine before you start protein synthesis?
Methionine (which is very important in 1 C metabolism)
You formylate it with Formyl THF
that NUCLEIC ACIDS (both RNA and DNA) often are ___________.
Methylated. This shows one nucleotide in the process of being methylated by SAM.
Draw the stucture of PRPP
Selenium can replace the sulfur in ________.
Glutathione Peroxidase contains a ___________.
What happens when animals have high amounts of selenium?
Cysteine
Selenocysteine
Trace amounts of Selenium are essential in the diet. Higher amounts of Selenium are quite toxic. Plants don’t have this enzyme and thus plants can live on Selenium deficient soil. In New Zealand the soil is Se deficient so the sheep got cardiovascular disease, until they figured it out and sprayed Se salts on the hay.
Heme groups are broken down to linear ____________.
What is Billirubin? And sketch it out.
What happens if you have Gilbert’s Syndrome?
Bilirubin is the pigment found in a bruise, or in jaundice. Sometimes people with sickle cell disease have yellow sclera, the whites of the eyes. That color is from Bilirubin.
People with Gilbert’s Syndrome can’t attach their bilirubin to Glucuronic acid and therefore always have higher bilirubin than people without the syndrome. If you have a hemolytic disease (like Sickle Cell) it can be a very bad thing to have Gilbert’s Syndrome.
Name the RNA and DNA bases along with thier nucleosides, and nucleotides
What is the drug Acyclovir? (Don’t need to know how to draw)
An antiviral drug used against Herpes, Chicken Pox, and Shingles. Similarly modified nucleosides are also used against HIV/AIDS.
What is the point of this image?
Did DNA exist before proteins?
If you were to learn more about nucleotide metabolism, you would see that everything happens at a specific phosphorylation state. The enzyme that produces 2’deoxynucleotides ONLY works on nucleoside DIPHOSPHATES.
There is no way that DNA existed before proteins. There was nothing but RNA until this enzyme came into existence, and LUCA had this enzyme.
Draw out the steps for Beta Oxidation of Fatty Acids
Draw the Purine Salvage Pathway.
The enzyme that reduces _______ is called Dihydrofolate Reductase (DHFR) and uses ________ as the cofactor.
If we can inhibit DHFR, we can stop DNA synthesis. When would we want to stop DNA Synthesis?
DHF, NDPH
Chemotherapy uses drugs that target cancer cells. And cancer cells like to divide and grow, divide and grow.
If we disable DNA synthesis, then maybe we can stop all of that cell division and kill the cancer cells.
Draw the cycle where Serine can provide the methylene in the little cycle that supports Thymidylate Synthase.
Methotrexate can block DHFR, and at the top we see that Fluorouracil can block Thymidylate Synthase.
Methotrexate matches the structure of _____/_____
THF,DHF
Draw the sequence of structures from AMP to Uric Acid.
Why is it dangerous when purine becomes detached from its sugar?
It can only be oxidized and the end product of oxidation is Uric Acid which is almost insoluble.
What does this image show about uric acid?
Uric Acid actually forms crystals inside cells and joints and these can cause physical damage.
Gout can cause exquisite pain in, say, the toe joints. The crystals form in areas of low blood flow.
Lesch Nyhan Syndrome causes severe psychological anomalies including forms of self mutilation. It is not water soluble.
Allopurinol is a drug which “gums up the works” and inhibits _______ _______.
Xanthine Oxidase
In protein chemistry, _________ and ___________are useful tools because they always cut up a protein in the same way so you can get a consistent set of fragments.
What do restriction enzymes allow us to do?
Why do bacteria make DNA cutting enzymes?
- Trypsin and Chymotrypsin
- Restriction Cleavage Enzymes allow us to do that with double stranded DNA. They are very precise and only cut at a certain sequence.
The sequence has to be a “palindrome” which in this case means that the 5’3’ sequence is the same on the top and bottom strand.
- It is self defense against viruses. Viral DNA will “hijack” the cell and take over. So it has to be cut up.
The bacteria’s OWN DNA is protected because the cleavage sites are methylated.
The names of the Restriction Cleavage Enzymes tell you what bacteria they are from, EcoRI is from ___ _____.
Why are lap joints useful?
E Coli
Lap joints are the most useful because they produce DNA fragments with “sticky ends.” The short single stranded parts are Watson-Crick base paired with each other.
What does this image show?
This shows Viral DNA cut up by 4 different restriction enzymes.
The fragments were separated by size, probably in an Agarose Gel, and visualized with Ethidium Bromide.
The patterns are strikingly different.
Why is only the LAST nucleotide in each chain above fluorescent, while all of the other nucleotides are non fluorescent?
DI-deoxynucleotides are chain terminators and these are also the ones made fluorescent.
Using colors this way allows the process to be highly automated. With graduate students staring at photographic films, you would never be able to sequence the human genome!
Draw and describe Dideoxy Analog.
The DNA chain grows when a new nucleotide is added to the 3’ hydroxyl.
2’3’ dideoxynucleotides “poison” this process because they have no 3’ hydroxyl.
What is an “Electrophoresis Ladder?”
How do you read it?
When you have run 4 separate experiments with each nucleotide as the ddNTP you can run an “electrophoresis ladder” which tells you the whole sequence.
Read up from the bottom, CGATC etc. The shortest fragments will be at the bottom and those will be near the start. On an exam just tell me the sequence you see and DO NOT figure out the other strand and write that.
What does this image show?
How does it work?
20 cycles =
30 cycles =
This shows the concept of PCR – you have primers that match the 2 strands before and after the gene you want amplified.
Then you cycle the temperature to separate the strands, and re-anneal. Each round of PCR gives you double the amount of “desirable” DNA
20 cycles = a million fold increase, 30 cycles = a billion.
Describe how PCR works in (more) deatail
If you know enough about a target region, say a certain gene, you will be able to make Primers that will mark the beginning and end of the gene.
Of course the 2 primers will be on different strands. You heat up the DNA (this is in vitro) so that the strands separate.
Then you cool the solution a little and the primers adhere, and DNA Pol synthesizes Watson Crick copies of each strand.
To facilitate the heating and cooling Taq DNA Pol is used – this is from a thermophile, Thermus Aquaticus, and works well at high temperatures.
What does this image show?
This is a forensic DNA test – the crime was rape and there are 2 suspects and the husband. The “Sperm DNA” column in the middle matches only Suspect 1.
What if the gene doesn’t have the right restriction sites?
Linkers or Polylinkers are man-made sequences that contain a variety of restriction cleavage sites. You can “glue” linkers onto the 2 ends of the gene and then cleave to get your sticky ends.
(Need to know plasmid Pbr322) Where would we want to put the added gene if we want to be able to tell which bacteria have the gene and which do not?
What happens when the good bacteria are dead?
Eco RI doesn’t tell us anything. But if we insert the gene at the Sal I site, then we will get bacteria which can be killed by Tetracycline.
This gives us a way to discriminate and tell which bacteria have the recombinant plasmid (instead of un-reacted plasmids).
SO you want to keep the bacteria that are killed by Tetracycline. BUT NOW THEY ARE DEAD?!?
Replica Plating is the answer, we have many identical agar plates so once we kill the “good” bacteria we can go find them on the replicas.
By the way we’ll also kill whatever bacteria we can with Ampicillin. But if Ampicillin kills them, we don’t want them, because they don’t have ANY plasmid inside.
How are plasmids used as vectors?
Plasmids are useful “vectors” for getting DNA inside of bacteria. Larger pieces of DNA can be put inside viruses like Phage Lambda.
(The book has more details about Phage Lambda, I just want you to know it is a viral “vector.”)
What is a YAC?
Yeast Artificial Chromosome. The general structure of yeast chromosomes are like those of other eukaryotes including us.
Centromere in the center and telomeres at both ends. ( I am not asking you to know how YAC’s are used in detail.)
What is a gene library?
A gene library is more than a giant pile of books, and you have to kind of use tricks to find the book you are looking for.
What is happening in this image?
Here the whole slide is covered with a “lawn” of bacteria (none of which have the gene we want) and we are infecting them with Phage Lambda, some of which DO have the gene we want.
So the “clones” make their spots by eating the bacteria they encounter. And then you can make a “blot” and look for the gene with a radioactive DNA probe.
What does this picture illustrate?
This just illustrates the human genome, and where some interesting enzymes are located on the chromosomes.
How are humans similar to mice?
Looking at the DNA it might be hard to tell. Mice have mostly the same DNA that we do, but the chromosomes are arranged differently.
This should be true of all mammals.
The Chimpanzee genome should look all but identical to ours.
How are printing presses related to DNA replication?
Up until about 600 years ago there was no such thing as a printing press, so all books had to be copied out by hand.
The monks would sometimes introduce spelling errors – that would correspond to mutations, and the copying would be Replication of DNA.
Replication is _______-_______. Describe it.
Each daughter cell gets one strand of Mom’s DNA and one new strand.
One new and one old in the daughter cell.
How does damaged DNA get repaired?
This chapter is also about repair processes. Both replication and repair involve DNA synthesis.
Get rid of damage after replication with repair.
How is Deinococcus radiodurans related to DNA repair?
It is an organism which can sustain heavy radiation damage and somehow put its genome back together. In the 1950’s researchers exposed canned hams to levels of radiation which should have destroyed all living cells. Later the meat spoiled and the organism was discovered. Some call it “Conan the Bacterium”! It’s related to Thermus Aquaticus.
Takes DNA pieces and the places it back together and sent to space and get “zapped” by cosmic rays. Don’t worry about getting damaged cause it puts itself back together.
What does Bacterial DNA Polymerase I do?
What is a Klenow Fragment?
Bacterial DNA Polymerase I (repairs) is remarkable, it has only a single subunit but it has 3 different activities.
This Klenow Fragment of DNA Pol I has both the DNA Polymerase site (which adds nucleotides to DNA) and a 3’ to 5’ exonuclease activity.
The DNA is grasped between the “thumb” and the “fingers” of the enzyme.
No DNA Polymerase enzyme can START a new strand of _____.
How do you build DNA?
DNA
No DNA Polymerase enzyme can START a new strand of DNA. To START building DNA you have to have an enzyme make a primer of RNA.
Primase is the name we give this RNA polymerase. Once the primer has been made, it can be lengthened by a DNA Polymerase, for example Pol I or Pol III.
What is helicase?
Helicase pulls apart the 2 strands of DNA so that replication can occur, it keeps the Replication Fork moving down the DNA.
There are two interesting homologies. 1) this six-fold structure is homologous with the 3 alpha 3 beta structure in the F1 ATPase of Mitochondria.
2) The purple structures in each subunit are P-loop ATPases. P-loop ATPases are found in G proteins and in ABC (ATP binding cassette) transporters.
Don’t confuse P-loop ATPase with P-type ATPase, that is a different kind of transporter and those do not have P-loops.
Myosin in muscle is dependent on a P-loop structure and of course the mitochondrial F1 ATPase also has P-loops.
What is the gamma “camshaft?”
In the mitochondrial F1 ATPase it is the gamma “camshaft” that runs through the middle of the hexameric structure.
In Helicase, shown here, it is one strand of DNA which is pulled through the center by conformational changes caused by ATP hydrolysis.
In the RNA chapter we will learn that the RHO protein is also hexameric, and that has RNA running through the center.
What type of plasmid is this?
This would be a “relaxed” plasmid with 25 twists and a linking number = +25.
Right handed twists are counted as positive numbers, but right handed writhes are negative.’
Unwinding – the same DNA goes down to +____.
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