Gene regulation IIBiochemistry 302February 27, 2006Molecular basis of inhibition of RNAP by Lac repressorCRP/DNA complex−35 promoter site−10 promoter siteLewis, M. et al. (1996) Science 271:1247−60Lehninger Principles of Biochemistry, 4th ed., Ch 28IPTG may serve to drive Lac repressor DNA-binding helices apartLewis, M. et al. (1996) Science 271:1247no IPTG+ IPTG (note change in position of dashed transparent helices) N-terminal subdomainof coreC-terminal subdomainof coreHinge helix region: an allosteric switch• no IPTG bound– Packed α-helical “hinge”– α-helices stabilized by DNA (operator) and core contacts• w/ IPTG bound– Rotation and translation of N-term subdomains– Destabilization of the hinge helices– Unpacking of hinge helix →headpiece “unfolding”– Loss of specific DNA contacts: concerted folding and binding in reverse– Repressor now has same low affinity for operator and non-operator sequencesConceptual models of allosteric changes that disrupt DNA-bindingRotation and translationFunctional groups in DNA that facilitate protein (TF) bindingGroups used for base-pair recognition are shown in red. Lehninger Principles of Biochemistry, 4th ed., Ch 2832322323Amino acid-base pair interactions often observed in DNA-protein binding• Amino acid side chains most often H-bonded to bases in DNA (Asn, Gln, Glu, Asp, Lys, Arg)• No simple amino acid base interaction code….• ….but some restricted interactions– Asn or Gln with N6and N7 of adenine – Arg with N7 and O6of guanine• DNA binding sequences cannot be inferred from protein structure.Lehninger Principles of Biochemistry, 4th ed., Ch 28Lac repressor HTH motif (~20 aa): Shape, surface complementarity (fit), and hydrophobic interactionsHTH motif is shown in red and orange. DNA is blue.Surface rendering of HTH motif (gray) bound to DNA (blue).Protein and DNA separated to highlight some groups interacting via hydrophobic (orange) or H-bond contact (red).Lehninger Principles of Biochemistry, 4th ed., Ch 28Catabolic operons: Regulation by multiple signals/ligands that target different TFsCRP 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 28Other 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 encoding enzymes needed for amino acid synthesis (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 → TrpTrp inducer Dimeric HTH proteinaporepressor(when trp levels are low)Secondary mechanism of repression: moderate to high Trp levelsFig. 26-33Structure(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 28Mechanism of transcriptional attenuationRibosome 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.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 28Another view of attenuation emphasizing importance of the ribosomeFig. 26-36Regulons: Network of operons with a common regulator• Metabolism of secondary sugars– Lactose, arabinose, and galactose– CRP-cAMP-dependent• Heat-shock gene system– Replacement of σ70specificity 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) σ70σ32Lehninger Principles of Biochemistry, 4th ed., Ch 28Induction of SOS response in E. coli(LexA-dependent regulon)• Cellular response to extensiveDNA damage• Induced genes mostly involved in DNA repair• Mechanism: Proteolytic inactivation of LexArepressor– RecA/ssDNA-dependent– Interaction of ssDNA-bound RecA stimulates intrinsic protease activity of LexA.– LexA 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