control of gene expression Flashcards
reasons for studying gene expression in E.coli
bacterial gene expression is target for some antibiotics
bacteria are key human pathogens
E.coli acts as host for recombinant DNA
transcription
transfer of information from dsDNA to ssRNA
transcription in E.coli
- promoter (upstream of transcribed region)
- transcribed region (polycistronic RNA allowing coordinated expression of group of genes)
- terminator
E.coli promoter
40-60 bp region
binding site for RNA polymerase
2 hexameric sequences at -35 and -10
E.coli promoter strength
dictated by sequence
dictates efficiency of transcription initiation
closer to consensus, stronger the promoter
E.coli RNA polymerase
Mg2+ dependent
multi sub-unit
core: 2a 1b 1b’ 1 w
catalyzes transcription
can’t bind to promoter
holoenzyme
core + sigma factor
sigma factor
binds to core > holoenzyme
directs recognition of promoter sequences
main sigma factor > sigma 70
sigma 70 gene
rpoN
alternative sigma factors
envelope stress
stationary phase
flagellar regulation
nitrogen assimilation
heat shock
iron metabolism
sigma70 nucleotide sequences
-35 TTGACA
-10 TATAAT
Elongation direction
5’-3’
txn speed in e.coli
20-50 nt/ sec at 37 C
RNAP proofreading
no exonuclease activity
error rate 1/10000
2 types of termination
factor-independent
rho-dependent
factor-independent termination
4-10 consecutive A-T base pairs
G+C rich region with palindromic sequence immediately preceding A-T base pair series
rho
6 identical sub-units
helicase that unwinds RNA-DNA/RNA-RNA duplexes
rho dependent termination power
ATP hydrolysis
Rho dependent termination process
- rho loads on to rho utilisation site (C-rich sequences)
- RNA pol pauses at termination site
- rho unwinds RNA DNA hybrid
- RNA pol, mRNA and rho released
when is txn regulated
at initiation
strategies for transcription initiation regulation
1.repression
activation
Lac operon
repression at initiation
RNAP + Sigma make contact w -35 and -10 elements to form closed complex
negative regulatory factors
strong promoter
activation at initiation
weak promoter
positive acting factors
activator protein binds to DNA/ contacts to compensate for weak promoter
lac operon
cluster of genes under single promoter control
constitutive promoter
not regulatory
on at set level
lac operon requirements
lactose presence
glucose absence
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
homotetramer
4 identical subunits associated
(not covalently bound)
acid dissociation constant for repressor
Ka=[repressor DNA]/([free repressor][free DNA])
lac operator Ka
2*10^13
dissociation constant effect on affinity
high dissociation constant= high affinity
lac operon inducer
allolactose
allolactose mechanism
binds to lac repressor , causing conformational change
DNA binding sub-units separate by 3.5A
operator affinity reduced by factor of 1000
cAMP
made from ATP via adenylate cyclase
glucose intracellular transport inhibits adenylate cyclase/ prevents cAMP accumulation
decreasing glucose conc effect on cAMP
cAMP accumulates and binds to CAP protein
CAP (catabolite activator protein)
cAMP receptor protein activating lac operon
glucose
no lactose
no residual txn
repressor blocks txn therefore no CAP binding
no glucose
no lactose
no txn
repressor blocks RNAP
glucose
lactose
little txn
CAP doesn’t activate
no glucose
lactose
txn
no repressor > CAP activation
vibrio cholerae
many genes under control of CAP
CAP mutants defective in intestinal colonisation
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
genetic code features
triplet code
non-overlapping code
degenerate code
universal
singlet number of combinations
4 (A,U,C,G)
doublet number of combinations
16 (4^2)
degenerate code
amino acids encoded by more than one codon
number of stop codons`
3
number of codons specifying amino acids
61
number of start codons
1
AUG
specifies Met
tRNAs
small nucleic acids of 70-90 nts
5’ monophosphate
modified bases (ribothymidine, pseudoridine, dihydrouridine, inosine)
tRNA secondary structure
D loop (dihydrouridine)
T loop (pseudoridine)
variable arm
anticodon loop
amino acid acceptor site
tRNA 3D structure
amino acid acceptor stem
3’ terminal nucleotide sequence -CCA
aminoacyl tRNA’s
tRNAs joined to amino acids
catalyzed by tRNA synthetases
2 step reaction of aminoacylation of tRNAs
- AMP addition to carboxyl group > aminoacyl adenylate
- aminoacyl adenylate reacts w uncharged tRNA > aminoacyl tRNA and AMP
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
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
aminoacyl tRNA synthetase proofreading
editing site on tRNA synthetases
acylation site rejects amino acids larger, editing site rejects smaller amino acids
flexible acceptor stem function
can move amino acid between activation and editing site
codon anticodon interactions
antiparallel pairing
tRNA’s recognise more than one codon
wobble hypothesis
first 2 bases of codon base-pair with anticodon
5’ anticodon base can form non-standard H bond with 3’ codon base
ribosome
large ribonucleoprotein complexes
RNA component rRNA
stages of translation
initiation (initiator factors/ tRNA)
elongation (elongation factors)
termination (stop codon/ release factors)
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
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
70S initiation complex components
A (amino acyl) site
P (peptidyl) site
E (exit) site
shine-dalgarno sequence
complementary to 3’ end of ssRNA
Base-pairing positions 30S ribosomal sub-unit on mRNA
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
elongation
delivery of aminoacyl tRNA to A site
peptide bond formation
translocation
elongation factors
- EF-Tu/EF-Ts +GTP
- EF-G +GTP
peptide bond formation
amino group of aminoacyl-tRNA attacks carbonyl group of ester linkage, forms peptide bond and released deacylated tRNA
Peptidyl transferase catalysis
23S rRNA
Termination
release factors interact w stop codons
RF1»_space; UAA/UAG
RF2»_space; UAA/UGA
RF3.GTP aids RF1/RF2
RRF/EF-G promote ribosome dissociation
