Gene regulation IIBiochemistry 302

Bob KelmFebruary 28, 2005

Catabolic operons: Regulation by multiple signals targeting different TFs

CRP homodimer (subunit Mr22,000) bound to DNA. cAMP“inducer” is in red. RNAP interaction domain is yellow.

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.

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

Trp inducer

Dimeric HTH protein

aporepressor(when trp levels are low) Secondary mechanism of

repression: moderate to high Trp levelsFig. 26-33

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.

Short leader peptide has no known cellular function. Its synthesis is merely an operon regulatory device.

Lehninger Principles of Biochemistry, 4th ed., Ch 28

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 base-pairing.

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 heat-shock gene promoters

• SOS response to DNA damage– LexA repressor– RecA protein (unique role)

σ70

σ32

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 (r-protein) 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