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M2M > M2M Unit 1 > Flashcards

M2M Unit 1 Flashcards

1
Q

function of aminoacyl tRNA synthetase

A

put the correct AA on the correct tRNA

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2
Q

during translation, the mRNA is decoded by what process

A

the anticodon loop of a cognate tRNA pairs with the 3 nucleotide in mRNA

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3
Q

basic elongation cycle: 3 truths and 1 false

A

tRNAs enter into the A site and leave from the E site
peptide bond formation results in the transfer of the growing peptide chain from the P site tRNA to the A site tRNA
translocation occurs after peptide bond is formed
FALSE TRANSLOCATION REQUIRES ATP HYDROLYSIS

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4
Q

homologous recombination depends on:

A

the availability of the sister chromatid

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5
Q

during normal ds break repair:

A

the repaired sequence is indistinguishable from the sequence prior to the break if homologous recombination is used

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6
Q

the decision to use NHEJ instead of HR depends on:

A

blocking resection of the DNA ends
the activity of ligase 4
time in the cell cycle
NOT BRCA2

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7
Q

microRNAs play a role in:

A

translational repression

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8
Q

the mRNA cap serves important roles in splicing, 3’ processing, nuclear export, and translation. the 1st enzymatic step in the addition of the cap to the newly synthesized mRNA is:

A

triphosphatase

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9
Q

the proliferating cell nuclear antigen (PCNA) is a key component of the eukaryotic replisome. What function does it perform?

A

acts as a clamp which encircles the DNA and binds the replisome to affect processive synthesis of DNA

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10
Q

nuclear receptors such as estrogen receptor are transcription factors that mediate expression of hormone-regulated genes. these nuclear receptors are proteins which contain 3 identifiable domains:

A

transactivation domain
DNA binding domain
hormone binding domain

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11
Q

what nuclear receptor domain contains a Zn finger structure critical for steroid receptor function?

A

DNA binding domain

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12
Q

the fidelity of DNA replication occurring at the DNA replication fork comes from the: 3 things

A

inherent accuracy of the active site of the replicative DNA polymerase to select the correct incoming dNTP
the 3’-5’ proofreading exonuclease of the DNA polymerase to excise an incorrectly inserted nucleotide added to the 3’ OH terminus
a DNA repair activity that follows the replication fork

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13
Q

what type of DNA repair activity follows the replication fork?

A

mismatch DNA repair

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14
Q

Cockayne syndrome, Xeroderma Pigmentosum, and Trichothiodystrophy are all deficient in which DNA repair pathway?

A

nucleotide excision repair NER

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15
Q

during the initial phase of translation in bac, what guides binding of the mRNA to the 30S subunit of the ribosome?

A

pairing of the shine-delgarno sequence with the 16S rRNA

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16
Q

during splicing of the pre-mRNA, how is the 5’ slice site recognized?

A

base pairing of the U1 snRNA

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17
Q

what type of atomic interaction plays the central role in formation and stability of the alpha helix and beta sheet structures?

A

hydrogen bonding

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18
Q

which AAs are used to create turns in secondary structures?

A

proline and glycine

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19
Q

beta-amyloid protein production occurs as a result of which enzymatic cleavage sequence?

A

beta-secretase followed by gamma-secretase action on beta-amyloid precursor protein

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20
Q

how does Swi/Snf complex remodel chromatin?

A

uses HTP hydrolysis to break histone-DNA contacts and move histone octamer down DNA

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21
Q

what is the major role of binding of the TATA box binding protein to the TATA box?

A

helps direct assembly of the preinitiation complex

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22
Q

in the Michaelis-Menton description of kinetics, Kcat describes:

A

the number of substrate molecs converted to product at saturating substrate conc’s

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23
Q

which bond is broken during ATP hydrolysis?

A

beta-gamma phosphoanhydride bond

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24
Q

what intracellular conc of ADP will provide the most negative delta G for hydrolysis of the ATP?

A

lowest ADP conc (LaChatlier’s principle)

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25
Q

the RNA pol2 transciption factor IIH includes:

A

XPB protein
BPD protein
Cdk7, a cyclin-dependent protein kinase
NOT TATA BINDING BOX PROTEIN

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26
Q

what happens during splicing of euk mRNA

A

an intron is removed and 2 exons are joined

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27
Q

what does a spliceosome recognize

A

GU at the beginning of an intron
AG at the end of an intron
A branch point residue
NOT 5’ UTR

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28
Q

normal peptide with 100 AAs, has a mutation, now it only has 20. 1-10 are normal, but 11-20 are different. what happened?

A

nucleotide deletion in the codon for AA 11

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29
Q

you prepare a solution of glucose in water at standard conditions. why doesn’t any of it break down?

A

steps in metabolism of glucose require Ea

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30
Q

high Energy bonds (4)

A 
ATP (phosphoanhydride)
phosphocreatinine P-N
acetyl CoA
phosphophenolpyruvate C-O-P
NOT PHOSPHOSERINE
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31
Q

what reagent is used in Western blot to help detect a specific macromolec that has been transferred from the electrophoretic gel to the blotting membrane?

A

antibody

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32
Q

exome sequencing experiments are designed to interrogate which parts of the genome?

A

protein-coding regions

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33
Q

patients with Lynch syndrome (hereditary nonpolyposis colon cancer syndrome) have defects in which DNA repair pathway?

A

mismatch DNA repair

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34
Q

3 true statements about chromatin remodeling:

A

the ability of transcription factors to bind DNA can be affected by chromatin structure

  • the SWI/SNF remodeling activity is an ATP-dependent ATPase that helps disrupt histone octomers
  • HATs are co-activators
  • NOT N-TERMINI OF HISTONES ARE RICH IN ARGININES (should be lysine)
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35
Q

what’s different between bac and euk RNA polymerases?

A

RNA polymerase is modified by phosphorylation of the CTD when it clears the promoter

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36
Q

3 similarities between bac and euk transcription polymerases

A

5’ end of primary transcript is a triphosphate

  • isomerization results in formation of an open complex at the promoter
  • when RNA polymerase encounters a block, an RNA endonuclease activity is stimulated
  • RNA synthesis is completely processive
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37
Q

what role does eIF4E play?

A

functions as a cap binding protein

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38
Q

what can accurately describe G1 phase

A

cell division and growth are coordinated in G1

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39
Q

during prokaryotic translation, recognition of the shine delgarno sequence in mRNA allows:

A

placement of the correct AUG at the P site

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40
Q

what does Avastin do to treat tumors?

A

inhibits formation of new blood vessels induced by the vascular endothelial growth factor (VEGF)

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41
Q

what kind of molec is Avastin?

A

humon monoclonal antibody

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42
Q

what enzyme is used for next generation DNA sequencing?

A

DNA polymerase

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43
Q

what is most easily analyzed by short read DNA sequencing

A

genotype

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44
Q

energy used to catalyze DNA joining by ligase comes from

A

ATP, to promote high-E phosphodiester formation

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45
Q

4 common types of post-translational protein modification

A

phosphorylation of threonine
methylation of Lysine
addition of sugars to asparagines
acetylation of lysine

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46
Q

alzheimer’s disease is thought to be caused by

A

misfolded proteins

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47
Q

control of transciption is combinatorial, meaning:

A

few transcription factors come together in different ways to control transcription of many genes

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48
Q

normal gene expression can be controlled by:

A

mRNA export from nucleus

efficiency in translation

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49
Q

sensor proteins in the DNA checkpoint pathway:

A

bind DNA with blocked replication forks

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50
Q

how do human somatic cells maintain their size

A

cells arrest in G1 phase until they are big enough

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51
Q

where does the phosphate attach for DNA and RNA building blocks in the ribose?

A

5’ carbon

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52
Q

short read and long read reading lengths:

A

100bp and 10,000 bp

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53
Q

Laws of Thermodynamics

A

1st- E is conserved

2nd- entropy is always increasing

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54
Q

7 forms of E

A 
Kinetic
Potential
radiant
thermal
mechanical
electric
chem
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55
Q

thermodynamic equations

A 
deltaG0= -RTlnKeq
deltaG= deltaH-TdeltaS 
deltaG= -nFE (redox)
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56
Q

Keq related to deltaG

A

deltaG is negative when Keq is >1

deltaG is 0 when Keq is 1

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57
Q

how can you make a nonspontaneous rxn favorable?

A

couple it with a spontaneous rxn

standard free E changes are additive

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58
Q

2 High E compounds:

A

lipids
carbs
both have excess electrons

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59
Q

Purine vs Pyrimidine

A

Pure As Gold AG

CUT the Py

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60
Q

deoxyribose vs ribose

A

2’ -H or 2’ -OH

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61
Q

nucleoside vs nucleotide

A 
nucleoside= sugar + base
nucleotide= sugar, base, + P
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62
Q

relative solubility rule

A

phosphate > nucleotide > nucleoside > pyrimidine > purine

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63
Q

Gout and Lesch-Nyhan Disease are related to:

A

excess insoluble purine build up in tissues and kidneys

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64
Q

chemistry of phosphodiester bonds:

A

3’ -OH on ribose bonds with 5’ phosphate

ends of DNA/RNA chains will either have an exposed 3’ -OH or an exposed 5’ Phosphate

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65
Q

Avery, McCloud, and McCarty’s exp:

A

est DNA as genetic material through pneumococcus exp:

S strain killed, R strain didn’t; DNA from heat-killed S cultured with R killed

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66
Q

Franklin and Wilkins exp:

A

x-ray diffraction suggesting helical structure

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67
Q

Watson and Crick:

A

discover definitive double helix structure

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68
Q

Chargaff’s Rule:

A

suggested base pairing

ratios of G:C and A:T were equal

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69
Q

3D model for DNA:

A

right-handed, antiparallel, double stranded helix
hydrophilic sugar phosphate backbone on outside
hydrophobic base pairing on inside
major and minor grooves
about 10 bp per helical turn

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70
Q

how are electrostatic repulsions neutralized in DNA backbone?

A
  • positively charged species in cell (Mg)
  • base pair linkages offer stability
  • adj base pair stacking, offering delocalization
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71
Q

what does salt, pH, length, and GC content do to DNA stability

A

salt inc stability and Tm
pH extremes dec stability (alter base ionization and H bonding)
length inc stability
high GC content inc stability (3 H bonds- more delocalization)

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72
Q

linear vs circular DNA

A

linear: large, segments can become supercoiled
Circular: shorter; bound to itself, so no supercoiling problem

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73
Q

5 chemical modifications of DNA bases

A 
methylation
deamination
depurination
UV light
alkylating agents
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74
Q

DNA methylation

A

typically CpG–> Met-CpG
catalyzed by DNA methyltransferase
80-90% human CpG sites are methylated

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75
Q

CpG island

A

GC rich area without methylation

less methylation = more transcriptional activity

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76
Q

DNA deamination

A

C loses an amine –> U

potentially changes CG pair to a TA pair

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77
Q

DNA depurination

A

hydrolysis of N-glycosidic bond to lose purine base
-OH replaces purine loss
weakens backbone

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78
Q

DNA UV light interaction

A

UV light can dimerize adjacent Thymines

distorts DNA helix and blocks replication enzymes

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79
Q

UV damage repaired by:

A

nucleotide exicision repair NER

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80
Q

DNA alkylating agents

A

nucleophilic attack of bases on nucleotides

mustard gas, cisplatin

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81
Q

DNA polymerization and nuceloside analogs used for drug therapy

A

nucleoside analogs block replication of virally infected cells by being incorporated into the replicating chain
analogs make DNA chains nonfunctional

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82
Q

when are more and less specific nucleoside analogs used, respectively

A

more specific- used against retroviruses

less specific- used against cancer

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83
Q

4 methods to attack DNA metabolism:

A

block synthesis of precursors
intercalation
covalently bind bp’s
attack topoisomerases

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84
Q

using DNA probe and Tm for diagnostic techniques:

A

complementary DNA strands have high Tm

use a probe of known sequence against unknown sequence to see if Tm is same or lower (mutation)

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85
Q

3 classes of RNA in human cell

A

Structural
regulatory
information-containing

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86
Q

4- structural RNA’s and func’s

A

rRNA- make up ribosomes
tRNA- move RNA
snRNA and snoRNA- splicing and other in-cell modifications

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87
Q

2 regulator RNA’s and func

A

miRNA and siRNA- downregulate gene expression

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88
Q

1 info-containing RNA and func

A

mRNA- to be translated into proteins

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89
Q

how does puromycin mimic amino-acyl tRNA to terminate translation

A

puromycin is an antibiotic; mimics the acceptor 3’ end of tRNA
it covalently attaches to polypep chain and prevents completion of translation

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90
Q

origin of replication

A

specific sequence recognized by binding proteins
usually multiple short repeats w/ AT rich streak
1 in prokaryotes, 100s per chromosome

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91
Q

origin binding proteins

A

bind to the origin and become part of the complex

recruit Pol3

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92
Q

helicases

A

catalyze breakage of H bonds to unwind helix

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93
Q

SSB protein

A

bind to melted strands of DNA to prevent re-annealing

esp important for okazaki fragments

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94
Q

primase

A

enzyme that catalyze the the addition of RNA primer to being replication

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95
Q

DNA Pol1

A

DISTRIBUTIVE- replaces RNA primers using:
DNA polymerase, 3-5’ exonuclease activity, and 5-3’ exonuclease activity
NO binding clamp- so it’s slow and distributive

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96
Q

DNA Pol3

A

PROCESSIVE- synthesizes DNA strand from its compliment

bound tightly to DNA via sliding clamp- works fast and is processive

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97
Q

common to Pol1 and Pol3

A

proofreading activity AKA 3-5’ exonuclease activity

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98
Q

DNA ligase

A

enzyme responsible for sealing okazaki fragments once RNA primers ahve been replaced by Pol1

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99
Q

Telomere

A

sequence at end of chromosomes; large repeated segments
progressively shorter w/ each replication
cell becomes destroyed when telomeres become too short

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100
Q

telomerase-

A

enzyme ensures telomeres never shorten in immortal cells (germ cells)
act as reverse transcriptases on DNA ends
REPRESSED in somatic cells
DE-REPRESSED in cancer cells

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101
Q

topoisomerase/gyrase

A

enzyme responsible for relieving torsional strain in DNA helix AHEAD of replication fork
(gyrase is specific to prokaryotes)

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102
Q

reverse transcriptase-

A

enzyme responsible for copying INTO DNA usually from RNA

can be endogenous or exogenous

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103
Q

how DNA polymerase creates phosphodiester bond

A

breaks off phosphodiester bond from dNTP and uses liberated E to bind remaining P group to OH group on previous nucleotide in the chain

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104
Q

Does DNA polymerase require an RNA primer?

A

YES

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105
Q

DNA synthesis leading vs lagging strand

A

leading: orign binding proteins bind; DNA melted; topoisomerases relieve tension; Pol3 elongates DNA; 2 semiconserved strands are annealed
lagging: fragments are sealed together with DNA ligase

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106
Q

“End Replication Problem”

A

lagging strand can’t be synthesized all the way because RNA primer can’t attach past the end of the DNA
leads to shortened telomeres
telomerase maintains chromosomal ends in germ and cancerous cells

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107
Q

3 criteria for cell to become cancerous:

A

mutation/mismatch in a gene with proliferation;
cell can’t fix the problem
self-destruction is repressed/inhibited

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108
Q

xeroderma pigmentosum and cockayne syndrome are 2:

A

inheritable diseases from defective DNA repair

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109
Q

4 sources of DNA damage:

A

thymine dimers
uracils in DNA
bulky chemical adducts
double stranded breaks

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110
Q

thymine dimers

A

NER
UV causes adj thymines to bond, causing kinks
can cause Pol3 to fall of and Pol2 to take over (lots of errors)

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111
Q

uracils in DNA

A

BER
cytosine deamination –> uracil
can cause replication, transcription, and recognition problems

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112
Q

bulky chemical adducts in DNA:

A

NER

similar to thymine dimers, but caused by toxic large molecs binding to DNA bases

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113
Q

double-stranded breaks in DNA:

A

HR or NHEJ

can potentially lose half a chromosome

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114
Q

process of mismatch repair

A

mismatch recognized shortly after synthesis
endonucleases clip on either side
exonuclease and helicase excise problem
DNA Pol3 replaces w/ correct sequence (sealed by ligase)

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115
Q

mismatch reconition in E coli vs humans

A

E coli- old strand is methylated, so endonucleases clip the un-methylated strand
humans- unknown mech

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116
Q

nucleotide excision repair NER

A

repairs more overt problems that alter helix

recognize, clip by endonucleases, excise affected part, replace by Pol1, ligate

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117
Q

NER recognition pathway needs:

A

transcription factor TF2H

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118
Q

2 kinds of NER

A

transcription-coupled NER: w/in gene being actively transcribed
global genome NER: not w/in gene actively being transcribed

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119
Q

how does DNA replication continue with lesions? 2 ways

A

lesion bypass polymerization: usually occurs when cell can’t fix all the thymine dimers
bypass polymerases: attach when DNA Pol3 is stopped and NER can’t occur; they add nucleotides without proofreading (high error rate)

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120
Q

DNA damage checkpoint maintaining genome stability

A

cell cycle is paused if damage is sensed; repair machinery is up-regulated; 2 kinds of protein kinases (ATR and ATM) amplify signal to phosphorylate Chk1 and Chk2 for repair, arrest, or death. Chk1 and 2 phosphorylate p53, which causes cell cycle arrest or death. Chk1 and 2 kinases ensure problems have been fixed;

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121
Q

mutations in DNA damage checkpoints

A

lead to genomic instability- cell can’t regulate itself and may proliferate into cancer
this checkpoint is first step in guarding against cancer

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122
Q

functions of ATR and ATM

A

both are protein kinases in DNA damage checkpoint
ATR is for stalled forks
ATM is for double stranded breaks

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123
Q

RNA polymerase reaction

A

unidirectional
catalyzes phosphodiester formation in 5’ to 3’ direction based on 3’ to 5’ template
need tripphosphate nucleotide for spontaneous rxn

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124
Q

5 steps in transcription cycle common to bac and euk RNA polymerases

A

INITIATION
1- RNA polymerase binds to promotor sequence on DNA in closed complex
2- polymerase melts DNA near transcription start site- form transcription bubble
3- polymerase catalyzes phosphodiester bond of two initial rNTPs
ELONGATION
4- polymerase adv 3’ to 5’ on template strand, melting DNA and linking rNTPs
TERMINATION
5-polymerase releases completed RNA and dissociates from DNA at transcription stop site

125
Q

4 cellular RNA polymerases and func’s

A

RNA pol1- makes rRNA
RNA pol2- makes mRNA, snRNA, miRNA
RNA pol3- makes tRNA, lncRNA
mitochondrial RNApol- makes mito RNA

126
Q

promoter:

A

where RNA polymerase binds

sequence of DNA upstream of the transcription start site that positively affects expression of gene

127
Q

4 consensus elements of promoter and func

A

TATA box- 30bp upstream TATA sequence
initiator- +1 in some but not all euk genes
promoter proximal elets- promoter DNA sequences 30-1000 bp upstream
enhancer elets- even farther upstream; acts through DNA looping

128
Q

TATA mutation consequences

A

TATA box binding protein helps assemble pre-initiation complex at the promoter, so a mutation would cause reduced expression of gene
ie beta-thalassemia with B-hemoglobin

129
Q

how does alpha-amanitin block transcription

A

toxic sub. found in death cap mushrooms; inhibits RNA Pol2; BINDING ITS BRIDGE SUBSTRUCTURE translocation down the DNA chain can’t happen

130
Q

how does rifampicin block transcription

A

broad-spectrum antibiotic; acts by binding the beta subunit of bac RNA polymerase, PLUGS UP THE EXIT CHAMBER so elongation can’t occur

131
Q

4 components in RNA polymerase 2 pre-initiation complex

A

RNA pol2
general transcription factors (TF2_)
promoter DNA
mediator

132
Q

mutations in TF2H subunit syndromes:

A

TF2H is a transcription factor that also repairs DNA damge
cockayne’s syndrome and trichothiodystrophy= problems w/ NER
xeroderma pigmentosum- light sensitivity; cancer susceptibility; abnormal neuro; unscheduled DNA synthesis

133
Q

3 major ways most pre-mRNA’s are processed:

A

capping- replace 5’triPhosphate w/ 7-methylguanosine

splicing- excision of introns and desgementation of exons

cleavage/polyA- cleavage at 3’ end past consensus sequence and polyA of cleaved site

NOTE these take place while RNA is still being made

134
Q

pre-mRNA vs mature mRNA

A

pre: 3’ phosphate intact; has introns; 3’ end is unmodified
mature: 7-methylguanosine cap; spliced out introns; polyA tail added to 3’ end

135
Q

4 funcs of 5’ mRNA cap

A

5’ end is resistant to exonucleases
helps with splicing and processing through cap-binding complex
translation factor eIF4E recongizes cap for ribosome transport
signals for mRNA to degrade when cap is removed

136
Q

3 rxns required to add a 5’ mRNA cap to pre-mRNA

A

1- cut off last Phosphate at the 5’ end
2- add GTP backwards via guanylyl transferase (losing 2 phosphate groups)
3- methylate 7-position of guanosine cap via SAM (s-adenosyl methionine)

137
Q

conserved intron and consensus sequence in polyA tail

A

5’ intron: GU
3’ intron: AG
consensus sequence: AAUAAA

138
Q

how does alternative splicing permit multiple proteins to be produced by splicing defects

A

5’ splice site (Identified by snRNA) is a part of the intron, so if it’s accidentally made unrecognizable then it will be translated and the mRNA will continue on, creating a significantly different protein

139
Q

disorders caused by splicing defects:

A

Marfan’s syndrome- disruption of the fibrillin gene; pts are tall and prone to aneurysms

abnormal splicing of CD44- predictor of tumor metastasis

140
Q

func of U1snRNA, U2snRNA, and U2AFsnRNA in splicing

A

U1- binds to the GU 5’ splice site of introns
U2- binds to A branch point
U2AF- binds to AG 3’ splice site in introns

141
Q

what does Lariat splicing mech do

A

uses the U1, U2, and U2AF splicing mech’s to link 2 exons and excise the intron complex

142
Q

what is 5’ UTR and 3’ UTR

A

5’- region between +1 and start codon on processed mRNA

3’- between stop codon until end of transcript, incl consensus seq and polyA tail

143
Q

2 rxns to make mature 3’ mRNA

A

1-recognize consensus seq at pre-mRNA’s 3’ end and cleave soon after sequence
2- polyA of free -OH at 3’ end

144
Q

how is 3’ pre-mRNA end processing related to transcription termination

A

cleavage and polyA continue during RNA polymerase, it could soon fall off or could continue making mRNA
polyA tail seems necessary for RNA polymerase complex to detach itself and terminate

145
Q

2 func’s of mRNA polyA tail

A

protection from degradation

export mRNA from nucleus

146
Q

alternative polyA sites used to make more than 1 protein in a single gene

A

2 forms of immunoglobulin M exist because transcription can continue past 1st polyA site and end on 2nd polyA site (heavy and light chains)

147
Q

what are DNA control elets

A 
DNA elets that act locally
transcription factors bind to regulated gene expression
TATA box/initiator sequence
promotor proximal elet
enhancers
148
Q

what are DNA transcription factors

A

proteins encoded by 1 gene that act on other genes to regulate their transcription
can activate/repress many genes

149
Q

promotor proximal elet vs enhancer

A

PPE- usually w/in 200 bps upstream of start site; about 20 bp long; bound by transcription factors that regulate transcription
enhancer- much farther upstream/downstream than promoters; similar size and func to promoters

150
Q

beta-thalassemia:

A

mild inherited anemia due to promoter mutation of b-globin gene, so less b-globin is produced

151
Q

gamma-delta-beta thalassemia

A

more serious anemia caused by a deletion in the control region for transcription of all globin genes, so loss of globin translation

152
Q

hemophilia b leydon:

A

x-linked disease affecting clotting
problem with promoter region of clotting protein gene
tends to improve partially at puberty

153
Q

fragile x syndrome

A

x-linked
mental retardatyion, atypical face development w/ enlarged testicles
caused by expansion of CGG count upstream of a gene that causes high rate of methylation and transcriptional silencing of that gene

154
Q

role of transcriptional activators and repressors

A

bind to DNA control elements or to other factors bound to control elements
increase/decrease rate of transcription of that gene

155
Q

2 classes of transcriptional activators/repressors

A

sequence specific binding proteins SSB- bind to control elets in DNA via alpha-helix insertion in major groove
co-factors: bind to SSBs- can increase or decrease transcription efficacy

156
Q

2 domains of SSBs

A

1st- DNA binding domain; binds to DNA target; highly structured and conserved
2nd- activation of domain; recruit other proteins (cofactors or GTFs) to bind and affect transcription

157
Q

4 families of SSBs, based on tertiary structure differences

A

homeodomain
Zn finger
basic leucine zipper
basic helix-loop-helix

158
Q

homeodomain proteins

A

SSB

helix-turn-helix structure; affect many genes at once

159
Q

zinc finger protein

A

largest family of SSB
includes estrogen and androgen receptors
finger made of 2 antiparallel beta sheets and alpha helix, held together by Zn ion
finger binds w/ DNA

160
Q

basic leucine zipper protein

A

SSB- chop sticks
hydrophobic residues
dimerizes to bind DNA

161
Q

basic helix loop helix protein

A

SSB

muscle group

162
Q

craniosynostosis

A

mutation in SSB- homeodomain that upregulates gene

premature skull closure

163
Q

androgen sensitivity syndrome

A

mutation in SSB- Zn finger androgen receptor
downregulates transcription of genes controlled by male androgens
feminization

164
Q

Waardenburg syndrome

A

mutation in SSB- basic helix loop helix protein

deafness, pigmentation defects

165
Q

how is cominatorial control used as a mech for controlling gene expression

A

many SSB proteins can dimerize, to make many DNA binding sequences within the same family of SSB’s (Zn finger w/ Zn finger)

166
Q

how does chromatin structure affect transcriptional control

A

DNA-dependent ATPases: disrupt histone octamers, opening chromatin and exposing it for binding via hydrolysis (Swi/Snf complex)

167
Q

2 factors that irreversibly modify histones through acetylation of N-terminus

A

HATs- acetylators, co-activators

HDACs- deacetylators, co-repressors

168
Q

current theory on HATs

A

pattern of histone acetylation recruits co-factors to affect increased transcription rather than directly affecting it themselves by charge

169
Q

leukemia and histone activity

A

haematopoietic (blood)

chromosomal translocations over- activating fusion proteins that alter HDAC/HAT activity

170
Q

rubinstein-taybi syndrome and Histone activity

A

growth and mental retardation; broad thumbs and toes; craniofacial dysmorphism
mutations in one copy of CREB binding protein gene, a HAT important in development

171
Q

activators/repressors interaction with general transcription machinery vs chromatin

A

bind to GTFs or RNA Pol2 complex to influence initiation or elongation of primary transcript

regulate accessibility of DNA on chromatin to Pol2 transcription apparatus (acetylation, phosphorylation, methylation, ubiquination)

172
Q

how is specificity achieved with transcriptional regulators

A

specificity depends on binding with specific DNA control elets
regulation depends on DNA-protein and protein-protein activation/repression
interactions affect conformation of DNA and change access to gene
control elets are combinatorial- mix and match monomers for control

173
Q

how are SSB proteins regulated

A 
alter TF-ligand binding conformation
regulate entry into nucleus/access to DNA
regulate amount of TF in cell
regulate DNA binding action of protein
phosphorylation/dephosphorylation of TF
174
Q

how are nuclear hormone receptors controlled

A

Zn finger DNA binding motif;
steroid hormone enters cell and binds to nuclear hormone receptor, leading to conf change, recruitment of coactivators/repressors, and entry into nucleus

175
Q

how does tamoxifen act in breast cancer therapy

A

nuclear hormone receptor activity:
acts as an antagonist to estrogen
doesn’t allow dimerization, and prevents transcriptional effects from estrogen receptors

176
Q

how is SSB protein regulated by nuclear entry

A

NF-kB is normally bound to IkB hiding the NLS, which holds it in the cytoplasm
if IkB is phosphorylated, it’s targeted for degradation; degrading IkB shows the NLS to migrate into the nucleus and affect transcription

177
Q

How does aspirin act on IkB?

A

it blocks IkB phosphorylation, blocking IkB degradation

and it is anti-inflammative

178
Q

how can amount of activators/repressors be regulated in a cell
and ACP gene as an example

A

specific genes can target the activators/repressors for degradation

ex. ACP gene targets beta-catenin via ubiquinin pathway and phosphorylation/degradation
(colon polyps caused by insufficient ACP activity)

179
Q

how can DNA binding activity of a SSB protein be inhibited via Id proteins

A

Id proteins have helix-loop-helix domain, but no basic domain so can’t bind to DNA
(HLH domain allows dimerization, basic allows DNA binding)
Id proteins heterodimerizing decrease efficiency of the helix loop helix proteins

180
Q

what is a protein modification that can affect SSB proteins with CREB protein

A

ligand binds G-protein, phosphorylates CREB protein, recruits CREB Binding Protein (HAT), which recruts RNA Pol2 which leads to transcription

no phosphorylation= no CREB mediated transcription

181
Q

4 mechanisms to control levels of gene expression

A

transcriptional regualtion
control of mRNA export from nucleus (cap and polyA tail)
control of mRNA degradation (siRNA action)
control of efficiency of translation (IRE/IRP at 5’ UTR)
control of progein degradation (ie ubiquitination)

182
Q

7 pieces of machinery that drive translation

A 
ribosomes
mRNA
tRNA
aminoacyl tRNA synthetase
initiation factors
elongation factors
release factors
183
Q

func of ribosomes in translation

A

site for mRNA translation into AAs

184
Q

func of mRNA in translation

A

transcribed and spliced DNA copy that provides the 3 base codons that code for an AA’s addition to a peptide chain

185
Q

func of tRNA in translation

A

small piece of RNA with an AA on its 3’ end

186
Q

func of aminoacyl tRNA synthetase

A

enzyme responsible for adding the correct AA to the tRNA with the correct anticodon by ATP hydrolysis

187
Q

func of initiation factors in translation

A

proteins needed to initiate ribosome assembly and mRNA translation

188
Q 
func of elongation factors in translation
ex. PTC and EF2
A

factos assoc with elongation ofa newly synthesized protein

ex. PTC catalyzes peptide bond via ATP coupled to tRNA
ex. EF2 uses GTP to move new peptide bond over in 3’ direction one codon

189
Q

func of release factors in translation

A

proteins taht bind to the stop codon can terminate polypeptide synthesis

190
Q

start codon and what AA it encodes

A

AUG codes for methionine

191
Q

missense, silent, and frameshift mutations in translation

A

missense- adds different AA
silent- different codon but same AA
frameshift- alters reading frame

192
Q

4 phases of translation

A

initiation
elongation
termination
ribosome recycling

193
Q

initiation of translation

A

2 subunits of ribosome brought together by a piece of mRNA

194
Q

elongation of translation

A

codons are being read and corresponding tRNA is brought to A site of large ribosomal subunit
tRNA is then showed to P site and peptide bond is formed using PTC
new AA on the chain is moved via elongation factors and GTP to E site and uncharged tRNA is released

195
Q

termination of translation

A

end of mRNA, release factors bind to stop codon and terminate protein synthesis

196
Q

ribosome recycling of translation

A

ribosome is dismantled after termination to translate another mRNA

197
Q

bac translation initiation

A

prokaryotes: use upstream shine-delgarno sequence to align mRNA start codon in P site of 30S subunit
IF1 and IF3 bind to 30S subunit
IF2 delivers formylmethionine to be 1st AA
GTP hydrolysis releases remaining initiation factors and binds 50S subunit
and next codon is placed in P site

198
Q

eukaryote translation initiation

A

eukaryotes: Kozak sequences in intons that help find start codon
pre-initiation complex becomes initiation complex, then it scans down mRNA to find AUG
Initiation factors break off and large ribosomal subunit joins to form full ribosome
(5’ UTR can affect initiation- stop or slow)

199
Q

what is cap independent initiation

A

viruses can bypass the need for a cap to translate proteins
some viruses produce a protease that cleaves eIF to shut down cap-dependent translation
the virus can translate its genes by using the IRES (internal ribosome entry site) on RNA

200
Q

what is interferon stimulation

A

when a virus invades a cell and the cell can’t protect itself, it releases interferon into the extracellular space to come into contact w/ neighboring cells that recognize the interferon and translate anitviral proteins to protect themselves

201
Q

what is mRNA editing

A

RNA code can be altered after it is made

it can be translated differently in different parts of the body (one gene being truncated in certain areas)

202
Q

what is rapamycin treatment

A

a drug that inhibits m-TOR protein translation

it phosphorylates 4E-BP so initiation complex can’t form

203
Q

what is eIF2-alpha phosphorylation

A

when interferon is recognized by a cell, it phosphorylates eIF2-a to effectively turn it off
the enzyme can no longer bring in tRNA for translation

204
Q

antibiotics and how they affect translation

A

streptomycin, tetracycline, erthromycin, chloramphenicol

they inhibit translation by interfering with ribosome via tRNA binding, elongation, and peptidyl transferase

205
Q

2 mechanisms to control iron regulation IRE and IRP

A

Iron response element IRE- RNA-stem loop structure in mRNAs that bind IRPs

Iron response binding proteins IRPs- bind iron and regulate expression of Ferritin and TFR

206
Q

low iron vs high iron

A

low iron: little will bind to IRE-BP, so it binds to IRE. this stops Ferritin production and stabilizes transferrin receptor mRNA

High: iron will bind to IRE-BP and not to IRE, thus Ferritin mRNA is translated and TFR mRNA is degraded

207
Q

function of ferritin and Transferrin receptor TFR

A

ferrin- sequesters iron (lowers free iron in cell)
TFR- transports iron into cell

low cell iron: up-regulate TFR translation and ferritin levels go down

208
Q

func groups of an AA

A

alpha C with NH2, COOH, H, and R group

209
Q

nonpolar/aliphatic R groups

A

MAGLIV

not very reactive; made of hydrocarbons

210
Q

aromatic R groups

A

FYW

211
Q

polar uncharged groups

A

SCTPQN

unbalanced electronegativities

212
Q

polar charged groups

positive and negative

A

positive: HRK (histidine, arginine, lysine)
basic with charged amines

negative: ED (glutamate, aspartamate)
acidic with resonant oxygens

213
Q

func of disulfide bonds in proteins

A

S-S from 2 proximal cysteine residues stabilize tertiary and quaternary structure

ex. insulin, kertain in hair, and ribonuclease

214
Q

post-translational covalent modification of AA sidechain

hydroxylation of proline

A

in collagen, stabilizes structures,
mediated by Vitamin C

Scurvy is Vit C deficiency, therefore underhydroxylated collagens (weak)

215
Q

post-translational covalent modification of AA sidechain

carboxylation of glutamate

A

on prothrombin,
mediated by Vitamin K
required for effective blood clotting
Vit K deficiency leads to improper clotting

Warfarin prevents glutamate carboxylation,
therefore is an anticoagulant

216
Q

post-translational covalent modification of AA sidechain

glycosylation of asparagine

A

of proteins on cell membranes and that are secreted
increases hydrophilicity

congenital disorder of glycosylation (CDG) has malfunctions in this mech

217
Q

post-translational covalent modification of AA sidechains

acetylation and deacetylation

A

of histones in gene regulation (HAT and HDAC)

cancer treatment can involve blocking HDACs

218
Q

post-translational covalent modification of AA sidechain

reversible phosphorylation

A 
via kinases (adds P) and phosphatases (removes P) 
affects signal transduction

Gleevec (tyrosine kinase inhibitor) is a cancer treatment- competitively inhibits bcr-abl kinase so substrate isn’t phosphorylated so tumor cell can’t proliferate via a abberant bcr-abl gene

219
Q

post-translational covalent modification of AA sidechain

ubiquitin

A

added to protein
signals them to be sent to the proteosome for destruction

multiple myeloma- velcade inhibits a proteosome that degrades good proteins

220
Q

3 covalent bonds that make up backbone of polypeptide chain

A

peptide bond C1-N; partial double bond character; rigid bond

alphaC - peptide C; free rotation

amide nitrogen - alphaC; free rotation

221
Q

2 models relating protein structure to function

A

AA sequence dictates structure–>function

1-lock and key- protein only fits specific substrate
2-induced fit- conformational change

222
Q

mutations in AA sequence effects

A

some are polymorphic- differences in non-essential AAs
mutations can alter hydrogen and sulfur bonding –> vastly different structure and function
collagen and keratin need their strength

223
Q

func of proteases

A

break down peptide bonds via hydrolysis

general: trypsin, chymotrypsin, pepsin (food digestion)
specific: help to activate precursor proteins to their active form (blood clotting factors are proteases that activate factors to yield clots after a trauma)

ex. angtiotensinogen cleaved by Renin to form Angiotensinogen 1, which is cleaved by ACE to Angiotensinogen 2, which is active hormone regulating bp

224
Q

h bonds’ role in secondary structure

A

weak, but numerous bonds between H and O
H bonds between amide N and carbonyl O are the driving force for secondary structure

alpha helix- H bonds every n and n+4
beta sheet- H between 2 chains

225
Q

2 major secondary structure types

A 
alpha helix- about 30% of all structures
ALL bio helices are right handed
all sidechians point outward
ex. hemoglobin
Thalassemia- disrupted alpha helices

beta sheet- about 30% of all structures
polypeptide chains held together by h bonds
anti-parallel are more common
ex. immunoglobulin or antibody

226
Q

2 special protein structures
turns and loops
triple helix

A

turns and loops to compact proteins (small glycine and kinked proline)

triple helix in collagen- massive H bonding with lots of hydroxylated proline and small glycine

227
Q

tertiary and quaternary structures

A

tertiary- spatial arrangement of polypeptide chain
globular (most, lipid or water soluble, diverse)
fibrous (long and either helices or sheets, insoluble, structure/protection)
N-terminus synthesized and folds first
proteins can assist in folding via chaperones

quaternary- multiple polypeptide chains to form 1 functional protein
ex. hemoglobin

228
Q

role of loops in protein structure and func

A

enable polypeptide chain to form structures
can interact with other proteins with loops
mostly proline and glycine

229
Q

how Kd represents binding strength

A

Kd is the dissociation constant

Kd= CONC OF LIGAND when 50% of ligands are bound

230
Q

how does heme enable myoglobin to bind oxygen

A

free or exposed Fe would be oxidized irreversibly, and the protein itself can’t bind O2
a heme group is isolated w/in protein to work w/o producing free radicals
myoglobin is the main oxygen storage protein

231
Q

carbon monoxide poisoning

A

CO has similar size and shape of O2
CO binds 200,000 x stronger than O2, so it outcompetes O2 binding
blocks myoglobin, hemoglobin, and mitochondrial cytochromes involved in oxidative phosphorylation

232
Q

why is hemoglobin a good O2 transporter

A

myoglobin binds too strongly
hemoglobin has 4 heme groups interacting in positive cooperativity
Tense state: low affinity (in lungs)
high pH: high O2 binding (in lungs)
relaxed state: high affinity (leaving lungs)
low pH: low O2 binding (in tissues)

233
Q

3 factors that cause protein denaturation

A

heat
pH
chemicals

234
Q

Ribonuclease Refolding Experiment conclusion

A

protein was denatured, but regained full function
all info needed to fold protein is in primary AA sequence
cell environment not always necessary for folding
protein doesn’t randomly explore all conformations (Levinthal’s paradox)

235
Q

2 classes of chaperones and their func

A

heat shock proteins (Hsp70)- induced at high T; binds to hydrophobic region to prevent aggregation, can help transport proteins across membranes in unfolded states

chaperonin- cap and two 7-subunit rings; binds to hydrophobic region of protein; uses ATP and conf change to partially fold protein so it can continue to final shape

236
Q

why protein disulfide isomerase and protein prolyl isomerases are sometimes needed for protein folding

A

disulfide isomerase- enzyme needs to correct incorrectly bonded free Cysteines

protein prolyl isomerase- reforms proline from trans to cis for proper folding

237
Q

4 diseases associated with protein misfolding

A

Alzheimer’s
Prion disease
Parkinson’s
amyloidosis

238
Q

Alzheimer’s disease w/ protein misfolding

A

normal AB-40 folds correctly
AB-42 misfolds and aggregates into amyloid plaques
leads to b-amyloid plaque and tau tangles

239
Q

prion diesease w/ protein misfolding

A

prion protein misfolds, causing other prions to misfold
altered from alpha helices to beta sheets
causes aggregation within amyloid plaques
leads to neuron loss and gliosis

240
Q

parkinson’s disease w/ protein misfolding

A

beta-synuclein misfolds into Lewy Bodies

241
Q

amyloidosis w/ protein misfolding

A

generalized protein misfolding in the rest of the body leading to variety of diseases

242
Q

3 ways to purify a protein

A

size- gel filtration chromatography
charge- ion exchange chromatography
ligand binding properties- affinity chromatography

243
Q

gel electrophoresis purpose and process

A

determines size of proteins
apply detergent- denature and uniformly coat proteins to be negative
use ladder, gel and electric field to separate

244
Q

mass spec

A

determines sequence of unknown protein by molec mass

245
Q

deman degradation

A

label and remove n-terminal AAs one at a time to identify

246
Q

Western blots

A

use immunology to identify proteins on gel
transfer proteins to membrane, react with primary antibody, wash all unbound things, react with fluorescent secondary antibody to detect
ex. identify HIV infection (patient serum is primary antibody against HIV proteins)

247
Q

palindromic double stranded DNA sequences

A

read the same forward and backward on compimentary strands 5’ to 3’

likely to be cut by restriction endonucleases

248
Q

southern blot

A

denature DNA then blotting the gel after electrophoresis onto a membrane that binds DNA
wash membrane with short label probe sequences of DNA or RNA to visualize

used when analyzing large quantity of DNA for particular sequences (diagnose genetic disease)

249
Q

restriction fragment length polymorphism (RFLP) used to diagnose disease

A

RFLP: ex. looking for HbS mutation in sickle cell (in a restriction site)
digest patient DNA with diagnostic restriction enzymes
electrophorese against normal genome
southern blot with P32 labeled b-globin gene; look for 1 long band (abnormal) or 2 shorter bands (normal)

250
Q

DNA fingerprinting for diagnoses

A

ex. paternal test
PCR with primers that surround variable number tandem repeat (VNTR) sequences
electrophoresis/detection of altered size of DNA fragment patterns
compare sample against target(s) for similarity match

251
Q

transferring DNA, RNA, and proteins from electrophoresis gel to membrane

A

Southern blot- DNA
Northern- RNA
Western- protein

252
Q

4 characteristics of hybridization probe that allow you to detect specific DNA sequences on a membrane

A

specific length- determines annealing T
specific sequence- binds to DNA of interest
something for visualization (radioactivity)
quantity- add enough to outcompete other strand of DNA in annealing to target strand

253
Q

3 classes of enzymes used in recombinant technology

A

DNA polymerases- DNA–>DNA
reverse transcriptase- RNA–> DNA
DNA ligase- join DNA fragments

254
Q

4 main steps repeated in PCR amplification

A

1- add termal stable DNA polymerase (Taq polymerase) and dNTPs and primers
2- denature DNA at 95 degrees
3- cool to 55 degrees for primer hybridization
4- warm to 72 degrees so polymerase copies the DNA

255
Q

PCR use for diagnosis

A

prepare a primer that hybridizes with a mutant copy of DNA
carry out PCR
signal will amplify if genetic material has mutation
ex. cystic fibrosis, beta-thalassemia, etc

256
Q

DNA sequencing

A

sequencing- use colored ddNTPs to stop synthesis and color last base; run through column to separate sizes; detector will read colors as they elute

257
Q

PCR vs DNA sequencing purpose

A

PCR amplifies a DNA segment; uses double stranded DNA

DNA sequencing- determines sequence of unknown fragment; uses ssDNA, uses ddNTPs

both use primer sequences to initiation gene replication

258
Q

6 cloning vectors

A 
plasmids
bacteriophage
cosmids
BAC
YAC
retroviral vectors
259
Q

plasmids-

A

vectors for amplifying DNA sequences in bac

simple but inefficient

260
Q

bacteriophage

A

vector used to infect E coli and use its replication machinery to produce the recombinant vector
more efficient than plasmid

261
Q

cosmid

A

hybrid of plasmid and bacteriophage

uses plasmid replication origin

262
Q

BAC

A

bacterial artificial chromosome

good for chromosome mapping and sequencing

263
Q

YAC

A

yeast artificial chromosomes

chromosome mapping and sequencing

264
Q

retroviral vectors

A

can carry very large inserts
introduce DNA to mammalian cells
delivers gene therapy

265
Q

use of mircoRNAs to measure mRNA levels

A

measure gene expression
they rely on hybridization, limited by background hybridization and signal saturation
can’t provide sequence level info
can miss mutations, modifications, and splice forms

266
Q

how do cells regulate size at the Restriction point

A

R point in G1 phase to check before S phase
somatic cells- make a decision whether hormones and GFs are present, and whether cell is large enough to undergo replication and mitosis
embryonic cells- R is bypassed; cells divide and get smaller
R point is 1st and most highly regulated checkpoint in cell cycle

267
Q

main goal of somatic cell cycle

A

to ensure exact duplication of the genome in S phase followed by exact division of genomes in M phase to produce identical daughter cells

268
Q

how do cells prevent re-replication of their genome in each phase

A

M- high CDK prevents building Pre-Replication Complex
G1- low CDK allows building of PRC
ORC proteins initiate replication by binding to the DNA binding origin and complexing with other proteins
S- high CDK activates replication and prevents PRC building

in short: pre-RC assembled during G1, activated during S, driven respectively by low and high CDK conc’s in the cell

269
Q

genetic instability causes in cell cycle

A

chromosome re-replication in S phase
or mis-segregation during mitosis

produces cancer or birth defects

270
Q

end point of mitosis

A

2 identical diploid cells from 1 diploid cell

271
Q

end point of meiosis

A

4 different haploid cells from 1 diploid cell
start: chromosomes A and a
1st- chromosome replication AA and aa
2- homologous recombination of chromosomes to provide genetic variation
3- separate homologs (meiosis 1: one cell AA and one cell aa)
4- separate chromosomes of daughter cells (diploid to haploid; 4 cells, A, A, a, a)

272
Q

differentiated, post-mitotic cells stuck in the R point

A

continue to grow without cycling

ex. neurons

273
Q

CDK’s role in cell cycle regulation

A

phosphorylate AA’s
are regulated by cyclin
Active CDKs are produced by growth-factor hormones and result in cell replication and duplication
found at particular levels in particular cells (elevated in a tumor is indicative of proliferation)
low levels of RB or highly phosphorylated/inactive RBs indicate high replication rates (inhibits entrance into S)

274
Q

CDI proteins

A

CDK inhibitors
turn off CDKs and repress cell cycle
mutations turn off the ability to turn off a CDK, leading to uncontrolled proliferation

275
Q

mitogen

A

signal protein that leads to activation of CDK4 (leads to positve feedback to increase proliferation)
produces cyclinD

276
Q

cell cycle checkpoint mechanisms

A

failing any checkpoint= cell cycle arrest
you can repair DNA at any phase (otherwise, apoptosis)
G1: R point; is cell big enough
S: should DNA be replicated?
G2: correct copies of new/old DNA?
M- did spindles form and act normally?

277
Q

enzyme and rxn rates

A

molec that increase rxn rate without being irreversibly changed
specific structures and active sites
some use specific cofactors or coenzymes
classified and named by rxn they catalyze

278
Q

Ea and reaction free E

A

Ea- E needed to overcome for reaction to proceed; dictates rxn rate
reaction free E- products - reactants E; dictates spontaneity

279
Q

how enzymes work

A

enzyme binds and stabilizes the substrate in T state (induced fit changes conf pushing it toward product)
this lowers Ea needed to form product

280
Q

3 types of active site chemistry to rearrange covalent bonds

A

metal ion chem- redox rxns
general acid-base catalysis- involves protons
covalent catalysis- transient covalent bond between enzyme and substrate

281
Q

cofactor vs coenzyme

A

cofactor- metal ions required for enzyme activity
coenzyme- organic ligand that binds to the enzyme allowing it to act on substrate
(prosthetic group- tightly bound ligand;
holoenzyme- complex w/ the enzyme
apoenzyme- enzyme dissociated from complex)

282
Q

Km vs Kcat

A 
Km= CONC of substrate at which rxn is 1/2 vmax; use lineweaver-burke plot to determine
Kcat= turnover number; rate constant for # substrate molecs converted per time under sat condictions

Kcat/Km tells overall enzyme efficiency
large ratio indicates very efficient

283
Q

4 types of enzyme inhibitors and func

A

competitive- inhibitor binds to substrate binding site
uncompetitive- inhibitor binds elsewhere; only binds to ES complex; lowers Vmax and changes Km
mixed/noncompetitive- inhibitor binds outside of active site to EITHER E or ES complex; affects both Km and Vmax
irreversible- permanent enzyme change (penicilin weakens bac cell walls)

284
Q

4 types of enzyme regulation pathways

A

allosteric regulation- molec binds to enzyme and causes conf change to allow/disallow activity
covalent modification of enzyme- phosphorylation (via Kinases)
regulatory protein binding- enzymes bound by proteins to activate/inactivate
proteolytic activation- enzymes are inactive until cleaved by another enzyme (trypisinogen –> trypsin)

285
Q

endogenous and exogenous dsDNA breaks

A 
meiosis
generation of immune receptor diversity
DNA replication nicks
insertion of retroviruses into genomic DNA
ionizing radiation damage
medical tests
286
Q

2 mechanisms of dsDNA break repar

A

NHEJ- can occur any time
doesn’t restore orignal DNA (cuts some out)
used with immune receptor diversity

HR- S, G2, and meiosis
goal is perfect repair using sister chromatid
used with everything else

both: repair damage from ionizing radiation

287
Q

NHEJ mechanism

A

Ku recognizes ds break
Ku recruits DNA-PKcs, recruits Artomis to form complex
nuclease to remove DNA if there’s damage
polymerase fills gaps
repair finishes with ligase
regulated by 53BPI

288
Q

HR mechanism

A

requires homologs
perfect repair- uses unbroken sister chromatid template to repair break
forms Holliday junction to repair breaks
junction is resolved to regenerate 2 ds DNAs
2 ways to cleave junc- results in cross over or non-crossover
regulated by BRCA1

in some cases of HR, use of other homolog as template (vs sister chromatid) can lead to LOSS OF HETEROZYGOSITY

289
Q

how do defects in ds break repair lead to to cancer risk

A

mis-repair leads to genetic instability

can lose signaling proteins (signals and transducers) and lose enzymes that mediate their repair

290
Q

3 classes of proteins that repair ds breaks

A 
sensing (Ku)
signal transudction (ATM)
DNA repair (HR)
291
Q

next-generation DNA sequencing-

short read sequencers

A

produce million-billion 100bp reads in a single run
low error rates (10-6)
commonly for high read-depth over specific subset of DNA template from complex mixture

292
Q

next-gen DNA sequencing

long read sequencers

A

produce about 10k reads of 10k+bps
high error rates (10-1)
useful in human genetics for linkking contiguous polymorphisms on same haplotype
single-molec approach to extract info from individual DNA template- “watches” single DNA polymerases
measures in close to real time- very long sequences can be generated

293
Q

next-gen data collection
read depth
error rate
contiguity

A

read depth AKA coverage- number of times a genome is sequenced (higher= high confidence)
error rate- quality of converting signal to observation
contiguity- linking variations with e/o, usually across long distances

294
Q

SNP identification criteria

A

ploidy need enough coverage to determine heterozygosity

295
Q

exome sequencing experiments

A

sequence the 1% of genome that codes for proteins

can identify novel coding SNPs among patients with same genetic disorder

296
Q

which types of diseases are best characterized by exome sequencing

A

single-gene Mendelian diseases
variants are RARE, and not likely to be in existing database of variations
variants are non-synonymous

eg. Miller syndrome- embryonic exposure to methotrexate

297
Q

prion structure normal vs infectious

A

normal- alpha helices

infectious- beta sheets; hydrophobic; clump into lesions

298
Q

how prion disease can be acquired

A

sporadic- random misfolding; infects other prions
inherited- familial strains; lead to early-onset of Creutzfeldt-Jakob Disease (fatal insomnia)
infectious- from diet or iatrogenetic (medical treatment)

299
Q

bovine spongioform encephalopathy vs variant creutzfeldt-jakob disease

A

BSE is mad cow disease

vCJD- strain of prion that originated in cows and led to human vCJD via injestion

300
Q

explain concept of prion strains

A

distinct characteristics- incubation time, clinical signs, distribution of protease resistant Prp in brains
all strains have same sequence as PrP

ex. hyper and drowsy strains tested in hamsters

301
Q

pathophysiology of Alzheimer’s

A

A-42 is incorrectly cleaved protein that incorrectly folds and becomes insoluble and leads to amyloid plaques
tau protein forms fibrillary tangles
tau and A plaques lead to inflammation and neuronal loss
occurs via post-translational amyloid precursor protein processing

302
Q

possible Alzheimer’s interventions

A

inhibit all APP products (which increase amyloid deposition)

use antibodies that attack A-42 (so it’s never misfolded)

303
Q

miRNAa

A

forms hairpin structure (double stranded)
don’t allow translation

bind to mRNA in cytoplasm
processed in nucleus by drosha and dicer
perfect fit- RNA degradation
imperfect fit- degradation or translational repression
degradation- RISC complex with argonaut to act on RNA (endonuclease)

304
Q

siRNA

A

from dsRNA cleaved by dicer, loads RISC complex- binds and cleaves; unwinding for recognition; cleavage
leads to:
chromatin remodeling (transcription silence)
regulation of transposons from viral infection
degradation

305
Q

piRNA

A

comes from ssRNA (no dicer)

regulates transposons with piwi and RISC

306
Q

lnRNA

A

lots of functions

regulates cardiac and skeletal muslces and immune response

307
Q

2 nc regulatory RNA therapeutics

A

potential to target any RNA thus any protein

RNAi- target specificity; target proteins that can’t be reached directly

antisense oligonucleotides ASOs- 4 types
focus on easy-access places (liver, eye)

308
Q

exon-skipping ASOs

A

use oligonucleotides to force a truncated exon to be skipped so cell will join other normal exons and put back into reading frame
works with muscular dystrophy

309
Q

anit-miRs and miRNA mimics

A

oligonucleotides used to antagonize or mimic miRNA
used to express something you don’t want being repressed
works with Hep C virus targeting the liver

M2M (4 decks)

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