Gene regulation II Biochemistry 302. Bob Kelm February 28, 2005

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1 Gene regulation II Biochemistry 302 Bob Kelm February 28, 2005

2 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 M r 22,000) bound to DNA. camp inducer is in red. RNAP interaction domain is yellow.

3 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)

4 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 Secondary mechanism of repression: moderate to high Trp levels

5 Structure(s) of the trp operon mrna leader (trpl) sequence (162 nt) Does the 3:4 pair structure remind you of anything?

6 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-tRNA Trp ] 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.

7 Another view of attenuation emphasizing importance of the ribosome Fig

8 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 σ 70 specificity factor by σ 32 RNA polymerase directed to different set of heatshock gene promoters SOS response to DNA damage LexA repressor RecA protein (unique role) σ 70 σ 32

9 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.

10 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.

11 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.