Gene regulation I Biochemistry 302 Bob Kelm February 25, 2005
Principles of gene regulation (cellular versus molecular level) Extracellular signals Chemical (e.g. hormones, growth factors) Environmental stress (e.g. temperature, nutrient availability) Intracellular effects Change in cell phenotype Specific changes in gene expression Seven processes affecting steady level of a protein Lehninger Principles of Biochemistry, 4th ed., Ch 28
Role of gene regulation in simple organisms vs metazoans Control of cell growth and division Bacteria: environment Lower eukaryotes (yeast): environment Higher eukaryotes (mammals): constant environment Specific chemical signals Cell:cell interaction Control of cell differentiation and development (metazoans) Mediated by precise genetic programs Generally irreversible (e.g. muscle differentiation) Endpoint may be death (e.g. rbc s, B-cells, skin cells) messenger The rate of synthesis and overall abundance of specific mrna transcripts may differ by over four orders of magnitude from cell type to cell type in metazoans.
Paradigms of gene regulation study of prokaryotic transcription factors Role of transcription factors Regulators of RNA Pol binding affinity Regulators of RNA Pol isomerization Regulation of transcription factors by ligands DNA-binding Protein-protein interaction Historical view (Jacob and Monod) RNAP + P K B affinity RNAP:Pc k 2 rate RNAP:Po elongating complex
Sugar utilization in prokaryotes: a paradigm of transcriptional regulation E. coli prefer glucose but can adapt to changes in nutrient availability Lactose, arabinose, maltose Metabolism of some sugars requires specialized gene products Jacob and Monod studying E. coli mutants displaying altered lactose metabolism, 1960s.. Predicted the existence of an unstable mrna template to account for rapid changes in β-gal activity Predicted the existence of a repressor that regulated the level of mrna by binding to a specific operator sequence on DNA Proposed unifying hypothesis of gene regulation where control of transcription initiation is key Minor transglycosylation product Lehninger Principles of Biochemistry, 4th ed., Ch 28
Structure of the lactose (lac) operon (structural genes + regulatory elements) Fig. 26-17 lacz: β-galactosidase (cleaves Lac Glc, Gal) lacy: β-galactoside permease (transport protein) laca: thiogalactoside transacetylase (modifies toxic non-hydrolyzable galactosides) laci (or lacr): Lac repressor (regulates the laczya cluster) CRP site: camp receptor protein (mediates regulation based on glucose levels)
Operon model based entirely on indirect evidence from genetic analysis Gal(β1 4)Glc high [glc] Gal(β1 6)Glc Primary control via repression! low [glc] high [lac] lactose or allolactose in E. coli Fig. 26-18 De-repression is necessary but not sufficient to activate the weak lac promoter.
Why the lac promoter is weak
Lac repressor can override CRP-induced activation. lac operon is co-regulated by changes in Lac repressor and CRP activator binding camp receptor protein (CRP) or catabolite activator protein (CAP) CRP-cAMP (inducer) complex binding to target site occurs when glucose is absent or very low. Fig. 26-21 Isopropyl β- thiogalactoside (IPTG) used in the lab Trans-activation not predicted by Jacob and Monod, also thought that the Lac repressor was RNA.
Mapping of protein binding sites in the lac regulatory region Note how operator elements are imperfect palindromes (inverted repeats) Binding affinity: O 1 (main operator) > O 2 or O 3 (secondary operators) Fig. 26-19
Lac repressor: an allosteric gene regulatory protein First regulatory protein shown to bind DNA Purified on the basis of IPTG affinity (Gilbert and Muller-Hill, 1966) Tetramer of four identical subunits ( 38 kda) Low intracellular concentration ( 10-8 M) Binds specifically to operator (K d = ~10-10 M) and non-specifically to DNA (K d = ~10-5 M). In response to inducer binding, repressor affinity changes from specific to non-specific mode. Three binding sites centered at +11 (O 1 ), 82 (O 3 ), and +432 (O 2 ) but only +11 and 82 are true operator elements w/ Lac forming a DNA loop between them.
Crystal structure of the Lac repressor (1996) provides insight into mechanism Lewis, M. et al. (1996) Science 271:1247 HTH Hinge N-terminal subdomain of core +11 82 93 bp loop Tetramerization helix: joins two dimeric units C-terminal subdomain of core T Helix Core domain: binds inducers e.g. IPTG, allolactose Headpiece: DNA-binding domain comprised of hinge & helix-turn-helix motif
Molecular basis of inhibition of RNAP by Lac repressor 35 promoter site 10 promoter site CRP/DNA complex 60 Lehninger Principles of Biochemistry, 4th ed., Ch 28 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 Packed α-helical hinge α-helices stabilized by DNA (operator) and core contacts w/ IPTG 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 Groups used for base-pair recognition are shown in red. Lehninger Principles of Biochemistry, 4th ed., Ch 28
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, 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. Lehninger Principles of Biochemistry, 4th ed., Ch 28
Lac repressor HTH motif (~20 aa): Shape, surface complementarity (fit), and hydrophobic interactions HTH motif is shown in red and orange. DNA is blue. Lehninger Principles of Biochemistry, 4th ed., Ch 28 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).