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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
constitutive promoter
not regulatory on at set level
26
lac operon requirements
lactose presence glucose absence
27
lac repressor
product of lacl gene key to lac operon regulation response to lactose 360 amino acid homotetramer binds to lac operator at 35bp palindrome
28
homotetramer
4 identical subunits associated (not covalently bound)
29
acid dissociation constant for repressor
Ka=[repressor DNA]/([free repressor][free DNA])
30
lac operator Ka
2*10^13
31
dissociation constant effect on affinity
high dissociation constant= high affinity
32
lac operon inducer
allolactose
33
allolactose mechanism
binds to lac repressor , causing conformational change DNA binding sub-units separate by 3.5A operator affinity reduced by factor of 1000
34
cAMP
made from ATP via adenylate cyclase glucose intracellular transport inhibits adenylate cyclase/ prevents cAMP accumulation
35
decreasing glucose conc effect on cAMP
cAMP accumulates and binds to CAP protein
36
CAP (catabolite activator protein)
cAMP receptor protein activating lac operon
37
glucose no lactose
no residual txn repressor blocks txn therefore no CAP binding
38
no glucose no lactose
no txn repressor blocks RNAP
39
glucose lactose
little txn CAP doesn't activate
40
no glucose lactose
txn no repressor > CAP activation
41
vibrio cholerae
many genes under control of CAP CAP mutants defective in intestinal colonisation
42
lac repressor exploitation for recombinant protein production
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
genetic code features
triplet code non-overlapping code degenerate code universal
44
singlet number of combinations
4 (A,U,C,G)
45
doublet number of combinations
16 (4^2)
46
degenerate code
amino acids encoded by more than one codon
47
number of stop codons`
3
48
number of codons specifying amino acids
61
49
number of start codons
1 AUG specifies Met
50
tRNAs
small nucleic acids of 70-90 nts 5' monophosphate modified bases (ribothymidine, pseudoridine, dihydrouridine, inosine)
51
tRNA secondary structure
D loop (dihydrouridine) T loop (pseudoridine) variable arm anticodon loop amino acid acceptor site
52
tRNA 3D structure
amino acid acceptor stem 3' terminal nucleotide sequence -CCA
53
aminoacyl tRNA's
tRNAs joined to amino acids catalyzed by tRNA synthetases
54
2 step reaction of aminoacylation of tRNAs
1. AMP addition to carboxyl group > aminoacyl adenylate 2. aminoacyl adenylate reacts w uncharged tRNA > aminoacyl tRNA and AMP
55
Aminoacyl tRNA synthetase classes
class 1 class 2 each class including enzymes specific to 10 of 20 amino acids each binds different faces of tRNA molecule
56
tRNA- ala identity element
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
aminoacyl tRNA synthetase proofreading
editing site on tRNA synthetases acylation site rejects amino acids larger, editing site rejects smaller amino acids
58
flexible acceptor stem function
can move amino acid between activation and editing site
59
codon anticodon interactions
antiparallel pairing tRNA's recognise more than one codon
60
wobble hypothesis
first 2 bases of codon base-pair with anticodon 5' anticodon base can form non-standard H bond with 3' codon base
61
ribosome
large ribonucleoprotein complexes RNA component rRNA
62
stages of translation
initiation (initiator factors/ tRNA) elongation (elongation factors) termination (stop codon/ release factors)
63
what does protein synthesis in bacteria start with?
fMet N-formylmethionine tRNA brings fMet initiatior tRNA charged w methionine and formyl group transferred by formyl transferase
64
initiation process
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
70S initiation complex components
A (amino acyl) site P (peptidyl) site E (exit) site
66
shine-dalgarno sequence
complementary to 3' end of ssRNA Base-pairing positions 30S ribosomal sub-unit on mRNA
67
initiation factors roles
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
elongation
delivery of aminoacyl tRNA to A site peptide bond formation translocation
69
elongation factors
1. EF-Tu/EF-Ts +GTP 2. EF-G +GTP
70
peptide bond formation
amino group of aminoacyl-tRNA attacks carbonyl group of ester linkage, forms peptide bond and released deacylated tRNA
71
Peptidyl transferase catalysis
23S rRNA
72
Termination
release factors interact w stop codons RF1 >> UAA/UAG RF2 >> UAA/UGA RF3.GTP aids RF1/RF2 RRF/EF-G promote ribosome dissociation

Biochemistry semester 1 (7 decks)

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