Gene regulation II Biochemistry 302 February 27, 2006
Molecular basis of inhibition of RNAP by Lac repressor 35 promoter site 10 promoter site CRP/DNA complex 60 Lewis, M. et al. (1996) Science 271:1247
IPTG may serve to drive Lac repressor DNA-binding helices apart N-terminal subdomain of core C-terminal subdomain of core Lewis, M. et al. (1996) Science 271:1247 no IPTG + IPTG (note change in position of dashed transparent helices)
Hinge 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 sequences
Conceptual models of allosteric changes that disrupt DNA-binding Rotation and translation
Functional groups in DNA that facilitate protein (TF) binding 3 2 3 2 2 3 2 3 Groups used for base-pair recognition are shown in red.
Amino 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 N 6 and N7 of adenine Arg with N7 and O 6 of guanine DNA binding sequences cannot be inferred from protein structure.
Lac repressor HTH motif (~20 aa): Shape, surface complementarity (fit), and hydrophobic interactions HTH 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).
Catabolic operons: Regulation by multiple signals/ligands that target 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.
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 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 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?
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.
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 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
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. LexA inactivates itself by catalyzing its own cleavage at a specific Arg-Gly bond in the middle of the protein.