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Biochemistry semester 1 > control of gene expression > Flashcards

control of gene expression Flashcards

1
Q

reasons for studying gene expression in E.coli

A

bacterial gene expression is target for some antibiotics
bacteria are key human pathogens
E.coli acts as host for recombinant DNA

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2
Q

transcription

A

transfer of information from dsDNA to ssRNA

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3
Q

transcription in E.coli

A
  1. promoter (upstream of transcribed region)
  2. transcribed region (polycistronic RNA allowing coordinated expression of group of genes)
  3. terminator
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4
Q

E.coli promoter

A

40-60 bp region
binding site for RNA polymerase
2 hexameric sequences at -35 and -10

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5
Q

E.coli promoter strength

A

dictated by sequence
dictates efficiency of transcription initiation
closer to consensus, stronger the promoter

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6
Q

E.coli RNA polymerase

A

Mg2+ dependent
multi sub-unit
core: 2a 1b 1b’ 1 w
catalyzes transcription
can’t bind to promoter

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7
Q

holoenzyme

A

core + sigma factor

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8
Q

sigma factor

A

binds to core > holoenzyme
directs recognition of promoter sequences
main sigma factor > sigma 70

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9
Q

sigma 70 gene

A

rpoN

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10
Q

alternative sigma factors

A

envelope stress
stationary phase
flagellar regulation
nitrogen assimilation
heat shock
iron metabolism

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11
Q

sigma70 nucleotide sequences

A

-35 TTGACA
-10 TATAAT

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12
Q

Elongation direction

A

5’-3’

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13
Q

txn speed in e.coli

A

20-50 nt/ sec at 37 C

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14
Q

RNAP proofreading

A

no exonuclease activity
error rate 1/10000

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15
Q

2 types of termination

A

factor-independent
rho-dependent

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16
Q

factor-independent termination

A

4-10 consecutive A-T base pairs
G+C rich region with palindromic sequence immediately preceding A-T base pair series

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17
Q

rho

A

6 identical sub-units
helicase that unwinds RNA-DNA/RNA-RNA duplexes

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18
Q

rho dependent termination power

A

ATP hydrolysis

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19
Q

Rho dependent termination process

A
  1. rho loads on to rho utilisation site (C-rich sequences)
  2. RNA pol pauses at termination site
  3. rho unwinds RNA DNA hybrid
  4. RNA pol, mRNA and rho released
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20
Q

when is txn regulated

A

at initiation

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21
Q

strategies for transcription initiation regulation

A

1.repression
activation
Lac operon

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22
Q

repression at initiation

A

RNAP + Sigma make contact w -35 and -10 elements to form closed complex
negative regulatory factors
strong promoter

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23
Q

activation at initiation

A

weak promoter
positive acting factors
activator protein binds to DNA/ contacts to compensate for weak promoter

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24
Q

lac operon

A

cluster of genes under single promoter control

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25
Q

constitutive promoter

A

not regulatory
on at set level

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26
Q

lac operon requirements

A

lactose presence
glucose absence

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27
Q

lac repressor

A

product of lacl gene
key to lac operon regulation response to lactose
360 amino acid
homotetramer
binds to lac operator at 35bp palindrome

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28
Q

homotetramer

A

4 identical subunits associated
(not covalently bound)

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29
Q

acid dissociation constant for repressor

A

Ka=[repressor DNA]/([free repressor][free DNA])

30
Q

lac operator Ka

A

2*10^13

31
Q

dissociation constant effect on affinity

A

high dissociation constant= high affinity

32
Q

lac operon inducer

A

allolactose

33
Q

allolactose mechanism

A

binds to lac repressor , causing conformational change
DNA binding sub-units separate by 3.5A
operator affinity reduced by factor of 1000

34
Q

cAMP

A

made from ATP via adenylate cyclase
glucose intracellular transport inhibits adenylate cyclase/ prevents cAMP accumulation

35
Q

decreasing glucose conc effect on cAMP

A

cAMP accumulates and binds to CAP protein

36
Q

CAP (catabolite activator protein)

A

cAMP receptor protein activating lac operon

37
Q

glucose
no lactose

A

no residual txn
repressor blocks txn therefore no CAP binding

38
Q

no glucose
no lactose

A

no txn
repressor blocks RNAP

39
Q

glucose
lactose

A

little txn
CAP doesn’t activate

40
Q

no glucose
lactose

A

txn
no repressor > CAP activation

41
Q

vibrio cholerae

A

many genes under control of CAP
CAP mutants defective in intestinal colonisation

42
Q

lac repressor exploitation for recombinant protein production

A

constant expression inhibits growth > low levels of desired protein
1. gene encoding protein controlled by lac repressor
2. grow cells
3. induce expression by IPTG addition mimicking allolactose

43
Q

genetic code features

A

triplet code
non-overlapping code
degenerate code
universal

44
Q

singlet number of combinations

A

4 (A,U,C,G)

45
Q

doublet number of combinations

A

16 (4^2)

46
Q

degenerate code

A

amino acids encoded by more than one codon

47
Q

number of stop codons`

A

3

48
Q

number of codons specifying amino acids

A

61

49
Q

number of start codons

A

1
AUG
specifies Met

50
Q

tRNAs

A

small nucleic acids of 70-90 nts
5’ monophosphate
modified bases (ribothymidine, pseudoridine, dihydrouridine, inosine)

51
Q

tRNA secondary structure

A

D loop (dihydrouridine)
T loop (pseudoridine)
variable arm
anticodon loop
amino acid acceptor site

52
Q

tRNA 3D structure

A

amino acid acceptor stem
3’ terminal nucleotide sequence -CCA

53
Q

aminoacyl tRNA’s

A

tRNAs joined to amino acids
catalyzed by tRNA synthetases

54
Q

2 step reaction of aminoacylation of tRNAs

A
  1. AMP addition to carboxyl group > aminoacyl adenylate
  2. aminoacyl adenylate reacts w uncharged tRNA > aminoacyl tRNA and AMP
55
Q

Aminoacyl tRNA synthetase classes

A

class 1
class 2
each class including enzymes specific to 10 of 20 amino acids
each binds different faces of tRNA molecule

56
Q

tRNA- ala identity element

A

single non-standard base pair
G3/ U70 mutation prevents aminoacylation with alanine
base pair deletion 14-65 doesn’t affect
» G-U base pair critical, rest dispensible

57
Q

aminoacyl tRNA synthetase proofreading

A

editing site on tRNA synthetases
acylation site rejects amino acids larger, editing site rejects smaller amino acids

58
Q

flexible acceptor stem function

A

can move amino acid between activation and editing site

59
Q

codon anticodon interactions

A

antiparallel pairing
tRNA’s recognise more than one codon

60
Q

wobble hypothesis

A

first 2 bases of codon base-pair with anticodon
5’ anticodon base can form non-standard H bond with 3’ codon base

61
Q

ribosome

A

large ribonucleoprotein complexes
RNA component rRNA

62
Q

stages of translation

A

initiation (initiator factors/ tRNA)
elongation (elongation factors)
termination (stop codon/ release factors)

63
Q

what does protein synthesis in bacteria start with?

A

fMet
N-formylmethionine
tRNA brings fMet
initiatior tRNA charged w methionine and formyl group transferred by formyl transferase

64
Q

initiation process

A

30S sub-unit binds to RBS/ shine-dalgarno sequence
initiator tRNA binds to start codon AUG
50S sub-unit binds to form 70S initiation complex

65
Q

70S initiation complex components

A

A (amino acyl) site
P (peptidyl) site
E (exit) site

66
Q

shine-dalgarno sequence

A

complementary to 3’ end of ssRNA
Base-pairing positions 30S ribosomal sub-unit on mRNA

67
Q

initiation factors roles

A

IF1/IF3 bind to free 30S sub-unit
IF2 complexes w GTP
30S sub-unit attaches to mRNA
charged initiator tRNA binds and releases IF3
50S sub-unit can then bind, displacing IF1/IF2 and GTP hydrolyzed

68
Q

elongation

A

delivery of aminoacyl tRNA to A site
peptide bond formation
translocation

69
Q

elongation factors

A
  1. EF-Tu/EF-Ts +GTP
  2. EF-G +GTP
70
Q

peptide bond formation

A

amino group of aminoacyl-tRNA attacks carbonyl group of ester linkage, forms peptide bond and released deacylated tRNA

71
Q

Peptidyl transferase catalysis

A

23S rRNA

72
Q

Termination

A

release factors interact w stop codons
RF1&raquo_space; UAA/UAG
RF2&raquo_space; UAA/UGA
RF3.GTP aids RF1/RF2
RRF/EF-G promote ribosome dissociation

Biochemistry semester 1 (7 decks)

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