High Yield (part II) Flashcards
nucleoside
base + sugar
could think ‘s’ in nucleoSide, stands for smaller!
nucleotide
base + sugar + phosphate
What is the linkage/bond that holds the sugar phosphate backbone (single strand) together?
phosphodiester bond -5’ end (phosphate) to 3’ end (OH)
What is Tm?
temperature required for denaturation of DNA -Tm is directly proportional to the number of G-C bonds
Zidovudine
Reverse transcriptase isn’t able to add nucleotide to 3’ end
- reverse transcriptase inhihbitor
- for treatment of HIV
-non targeted cancer treatment (injures body cells too!) basically prevents thymine from being synthesized -without one of its precursors DNA can’t be made -this should stop cell division and slow the growth of the cancer
5-FU
-antibiotic -binds to prokaryotic robosomal subunit (50S) -but since mitochondrial ribosomes are similar to bacterial, the drug can affect our mitochondria
Azithromycin
topoisomerase
breaks / rejoins phosphodiester bonds to relieve supercoiling - i.e. cuts strands (single strand - type I or double - type II)
DNA polymerase III
adds deoxyribonucteotides in the 5’ to 3’ direction (proof reads) -can also proof read in 3’ to 5’ (exonuclease) -makes 2 new strands leading/lagging (semiconservative replication
name for the strand of DNA that gets transcribed
template strand
name for the strand of DNA that is identical to the mRNA synthesized, except with T replaced by U
coding strand
Beta-thalassemia
decreased number of Hb molecules due to a mutation causing decreased transcription of the gene
Many antibiotics work by inhibiting bacterial mRNA synthesis. Rifamycin, for example, blocks transcription in E. coli. How is it possible that this drug is such a potent antibiotic to E. coli, yet does not cause harm to the person taking it?
- Could be due to difference in RNA polymerase
- Or Ribosome has different subunits in prok and eukary
- Could block the sigma factor
- **Sigma factor, polymerases are different, and ribosomes are different (anything that targets the difference in bacteria and us)
What would prevent the production of a normal transcript in bacteria?
Bacteria: Lack of sigma factor, lack of Rho factor, problem with RNA polymerase,
Both: (TATA box or promoter, lack of nuceotides, lack of ATP
What would prevent the production of a normal transcript in humans (eukaryotes)?
Euk: failure to splice, poly A tail or cap failure, problem unwinding DNA (issue with histones), lack of specific transcription factors (ones that enhance transcription)
Types of mutations?
point mutation - single base change
silent mutation - change results in same AA
missense mutation - specifies a different amino acid
non-sense mutation - produces a stop codon
insertion/deletion - results in frameshift mutation unless an entire codon is inserted or deleted
How can we convert euchromatin to heterochromatin?
HDAC (removes the acetyl group)
Determine if the regulation of gene expression is found only in eukaryotes (E), only in prokaryotes (P), or in both (B).
Chromatin remodeling Initiation of transcription Gene-specific TF Repression of an operon Attenuation of transcription mRNA processing Phosphorylation of eIF2α
Chromatin remodeling (E) Initiation of transcription (B) Gene-specific TF (E) Repression of an operon (P) Attenuation of transcription (P) mRNA processing (E) Phosphorylation of eIF2α (E)
what technique could we use for forensic analysis or paternity test?
DNA PCR of STR
- note: most the the DNA between any two people is identical (so how would this work? - STR (there are some difference - called short tandem repeats)
- so we’d have to use DNA recombination to isolate these STR portion of the DNA
what is Recombinant DNA and what can we use it for?
Joining DNA sequences into new combinations
-use it to
identify
amplify (PCR)
analyze
Identifying polymorphisms to diagnose disease
Polymorphisms = single nucleotide change in DNA
- Use a disease specific probe + normal probe
- child 1 has both normal, child 2 has both F508 - Restriction enzymes to cut DNA (has to be a restriction enzyme site present in the region that contains the polymorphism and if the gene is normal the restriction enzyme will cut there. But if polymorphism is present the enzyme will not cut the DNA
What is the purpose of a 2D gel?
Discovery technique to analyze protein expression
but its like trying to find a needle in one hay stack that isn’t in the other hay stack)
Separate proteins by isoelectric point (separating proteins by charge)
Then placing the tube horizontally on the gen (then top to bottom they separate by size)
how could we look at mRNA expression without using a northern blot? (because that involves a radio active label - fuck that we don’t want that)
Use reverse transcriptase to convert the mRNA to DNA, you can tell how much mRNA is present based on the amount of DNA you get
*only create cDNA library (RT transcribes coding regions only!)
*Can you use RT-PCR to determine viral infection?
YEP. just need one copy of the virus
-so get a blood sample to determine if someone is infected with *HIV
*what are microarrays used for?
*great for analyzing GENE EXPRESSION
she loves microarrarys
any question involving what to use for analyzing gene expression this will surely be the answer
*Are recombinant DNA techniques used to produce therapeutic compounds?
ah YEAH. today we use e. coli to make insulin
*ATP hydrolysis coupled to change in protein conformation.
Mechanical work: muscle contraction
only type that ATP physically participates in the reaction itself (adds a phosphate to the reactant.
Biochemical work: glucose to G6P (its phosphorylation, so the enzymes will be kinases)
What are the 2 regulated steps in the investment phase of glycolysis, and why are they regulated?
(uses 2 ATP)
conversion of glucose to G6P (currency of cell!) -hexokinase is the enzyme that accomplishes phosphorylation conversion of fructose 6 phosphate to fructose 1,6 bisphosphate -phosphofructokinase enzyme -they are regulated because they require ATP (phosphorylation), dont want to run the step and waste energy if they are not needed.
What are the 2 regulated steps in the payoff phase of glycolysis, and why are they regulated?
(4 ATP, net of 2 ATP 2 NAD+ are reduced)
conversion of 1,3-Bisphospho-glycerate to 3-Phospho-glycerate is regulated (bc it requires ATP!) -enzyme: phosphoglycerokinase conversion of Phosphoenol-pyruvate (PEP) to Pyruvate is regulated (bc it requires ATP!) -enzym: *pyruvate kinase
What does the Malate shuttle do?
delivers electrons to the mitochondria
examples of molecules that stimulate glycolysis?
-things in high concentration when energy is depleted will stimulate glycolysis ↑ [AMP]* ↑ [fructose-2,6-bisP] ↑ [fructose-1,6-bisP]
examples of molecules that inhibit glycolysis?
-a build up of the products of glycolysis would inhibit glycolysis ↑ [ATP]* ↑ [citrate] ↑ [glucose-6-P]** ↑ [NADH] ↑ [acetyl-coA] ↑ [alanine]
What would be the result of a partial Pyruvate Kinase deficiency?
affects RBC survival (type of anemia)
complete deficency is lethal
Glucose is at the center of / can feed into how many pathways?
glycogen path
pentose phosphate path
TCA cycle
fructose/lactose
lactate
glycosaminoglycans
examples of glycolysis intermediates used in other pathways
Used in protein synthesis, TG synthesis, AA synthesis
Predict the effect of elevated levels of each of the following on ATP production by glycolysis:
AMP
acetyl-coA
glucose-6-phosphate
NADH
fructose-1,6-bisphosphate
AMP: stimulate
acetyl-coA: inhibit
glucose-6-phosphate: inhibit
NADH: inhibit
fructose-1,6-bisphosphate: stimulates
SO, after glycolysis, how do we get to aerobic metabolism, what is the linker step?
- pyruvate molecules are converted to Acetyl CoA by:
- Pyruvate dehydrogenase complex (PDC)
- Now Acetyl-coA can enter the TCA cycle in the mitochondrial matrix
- PDC also produces 2 NADH!!
Pyruvate is only one source of Acetyl CoA, what other fuels can be oxidized to generate Acetyl CoA?
FAs
Ketone bodies
Amino Acids
Acetate
TCA cycle intermediates can be used in other pathways for?
AA synthesis (oxaloacetate, alph-ketoglutarate)
FA synthesis (citrate)
Gluconeogenesis (malate)
Heme synthesis (succinyl CoA)
Anaplerotic reactions
replenish TCA cycle intermediates
so the reverse of using intermediate to make other stuff when we have a bunch, is to synthesis intermediates
A 3 year-old male is brought to the pediatrician by his mother, who is concerned about what appears to be a regression in her son’s physical abilities involving movement and coordination. She reports that he seems to be weaker than he was 6 months ago, and that he occasionally has “jerky” movements of his limbs. Blood tests reveal moderate lactic acidemia. What deficiency could explain these findings?
- For some reason he’s not getting enough oxygen and therefore using glycolysis and producing lactic acid
- Could be some type of TCA deficiency (not getting enough of one of the enzymes)
- PDC deficiency (so we aren’t converting pyruvate into acetyl CoA (pyruvate accumulates and we see an increase in lactate)
* 3 is most likely - Increase in lactate is causing the symptoms (hes not getting enough Oxygen his muscles need to grow and function properly
This is a lethal condition
Movement of H+ leads to conformational changes in ATP synthase
Rotation on the C1-C5 part will cause conformational change in (F1) head piece
note: rotation of F0 has been proven experimentally
Other than thermogenin, what is another way to chemical uncouple the membrane (in other words ruin the H+ gradient)?
drugs/agents that disturpt the function of the ETC
(interfer with the ability of the complexes to transfer electrons)
rotenome
amytal
Carbon monoxide
CN (cyanide)
–How does her iron deficiency contribute to her fatigue and weakness?
complexes of the ETC need Iron (and copper)
to work
also her blood O2 is low and we need O2 as the final electron acceptor
*both of these contribute to impairing oxidative phosphorylation
A 55 year-old male presents to the emergency department with crushing chest pain, shortness of breath, and fatigue. An abnormal EKG and elevated cardiac enzymes confirm the suspected diagnosis.
In this patient’s heart tissue, what would you predict to find in terms of:
O2 level
[ATP]
[H+]
[Na+]
O2 level
-decreased because of blocked blood flow
[ATP]
-decresed because glycolysis is our only source of ATP
[H+]
-increased, due to the increase in lactate (from glycolysis)
[Na+]
-increased, Na+/K+ pump (requires ATP) so it isn’t pumping/functioning well
A 63 year-old patient is brought to the ER with lightheadedness, dizziness, and a headache. He has shortness of breath and seems confused. The neighbor that brought him in said that the patient is a heavy smoker, and that when he found him he was smoking in a poorly ventilated room.
In terms of aerobic metabolism, how is the suspected overexposure to cyanide causing the observed signs and symptoms?
CN (cyanide from cigarette smoke)
affecting complex 4
- the result is:
- decreased H+ gradient, which causes decreased ATP synthesis* (know this)
