Download Gene regulation I Biochemistry 302

Gene regulation II
Biochemistry 302
Bob Kelm
February 28, 2005
Catabolic operons: Regulation by
multiple signals targeting different TFs
Catabolite repression: Activity of lac operon
is restricted when both glucose and lactose
are present. E. coli would prefer to metabolize
glucose directly (via glycolysis) rather than
generating it from secondary sugars.
CRP homodimer (subunit Mr
22,000) bound to DNA. cAMP
“inducer” is in red. RNAP
interaction domain is yellow.
Lehninger Principles of Biochemistry, 4th ed., Ch 28
Other side of the coin: the biosynthetic
trp operon
• Amino acid biosynthesis consumes energy
– Advantageous to inhibit synthesis of biosynthetic
enzymes when end product (amino acid) is available.
– Regulatory goal is to repress gene activity.
• E. coli trp operon (in contrast to lac)
– Trp repressor is activated by ligand (Trp) binding.
– Additional regulation by premature termination of
transcription (attenuation – regulatory dimmer switch
involves ribosome positioning on 5′ mRNA)
• Discovered by Charles Yanofsky, common to many
biosynthetic operons including Trp, Leu, and His
• Dictated by changes in RNA secondary structure
• Extends the possible range of transcription rates
(moderate to high Trp levels)
Schematic of the E. coli trp operon
(regulation by Trp-induced repression)
chorismic acid → Trp
Dimeric HTH
protein
Trp inducer
aporepressor
(when trp levels
are low)
Fig. 26-33
Secondary mechanism of
repression: moderate to
high Trp levels
Structure(s) of the trp operon mRNA
leader (trpL) sequence (162 nt)
Does the 3:4 pair
structure remind
you of anything?
Lehninger Principles of Biochemistry, 4th ed., Ch 28
Mechanism of transcriptional attenuation
Ribosome follows
closely behind RNAP as
transcription proceeds.
The ribosome sterically
hinders 2:3 base-pairing
upon encountering
leader peptide stop
codon.
Ribosome stalling at
Trp codons due to low
[Trp-tRNATrp] i.e. when
Trp levels are low. This
allows more favored
2:3 base-pairing at the
expense of 3:4 basepairing.
Short leader peptide
has no known cellular
function. Its synthesis
is merely an operon
regulatory device.
Lehninger Principles of Biochemistry, 4th ed., Ch 28
Another view of attenuation emphasizing
importance of the ribosome
Fig. 26-36
Regulons: Network of operons with a
common regulator
•
Metabolism of secondary
sugars
– Lactose, arabinose, and
galactose
– CRP-cAMP-dependent
•
Heat-shock gene system
– Replacement of σ70
specificity factor by σ32
– RNA polymerase directed
to different set of heatshock gene promoters
•
σ70
σ32
SOS response to DNA
damage
– LexA repressor
– RecA protein (unique role)
Lehninger Principles of Biochemistry, 4th ed., Ch 28
Induction of SOS response in E. coli
(LexA-dependent regulon)
•
•
•
Cellular response to
extensive DNA damage
Induced genes mostly
involved in DNA repair
Mechanism: Proteolytic
inactivation of LexA
repressor
– RecA/ssDNA-dependent
– Interaction of ssDNA-bound
RecA stimulates intrinsic
protease activity of LexA.
– LecA inactivates itself by
catalyzing its own cleavage
at a specific Arg-Gly bond
in the middle of the protein.
Lehninger Principles of Biochemistry, 4th ed., Ch 28
Translation regulation in bacteria:
feedback control of ribosomal proteins
•
Translational feedback in
some ribosomal protein (rprotein) operon transcripts
– β operon contains genes
encoding RNAP subunits
– str operon contain genes
encoding translational
elongation factors
•
Specific r-proteins possess
both rRNA & operon-specific
mRNA-binding affinity
– Repress translation of
operon transcripts when
level of r-protein > rRNA
– Ensures balanced r-protein
and rRNA synthesis
Differential binding affinity of L10, S7,
S4, L4, and S8 for rRNA (higher) and
its owns mRNA transcript (lower)
makes this mechanism possible.
rRNA synthesis is also regulated by a
translation-dependent pathway
•
•
Stringent response: regulation
coordinated with [amino acid]
Amino acid starvation halts
rRNA synthesis by a sequence
of events triggered by binding
of an uncharged tRNA to
ribosome A site then….
– Stringent factor (RelA) binds to
ribosome
– RelA catalyzes addition of
pyrophosphate to 3′ position of
GTP then phosphohydrolase
removes one phosphate →
guanosine tetraphosphate
– ppGpp binds to RNA polymerase
and alters promoter selectivity
(including seven rRNA operons)
cAMP and ppGpp are major cellular
second messengers in E. coli.
Lehninger Principles of Biochemistry, 4th ed., Ch 28