Initiation of translation in eukaryotic cells:connecting the head and tail GCCRCCAUGG 1: Multiple initiation factors with distinct biochemical roles (linking, tethering, recruiting, and scanning) 2: 5 and 3 ends of mrna linked together and tethered to 40S subunit via eif/pabp complex. Longer poly (A) tract more efficient translation 3: Identification of start AUG achieved by scanning mechanism involving eif4b and eif4f(complex of 4E, 4A, and 4G). Initiator trna is Met-tRNA Met.
Recoding of internal UGA codon as Sec Sec-tRNA Sec (specific trna) eef Sec or SelB (specific elongation factor) SECIS (special recognition element in mrna) specific mrna hairpin 3 of internal UGA SBP2 (SECIS binding protein 2) ~94 kda adapter protein can form quaternary complex w/ eef Sec :GTP:Sec-tRNA Sec can bind ribosomes and 28S rrna ribosomal protein L30 (rpl30)? enhances incorporation of Sec in transfected cells competes with SBP for interaction with SECIS Models from S. A. Kinzy et al. Nucleic Acids Res. 33, 5172 (2005); M. Leibundgut et al. EMBO J. 24, 11 (2005)
Gene regulation I Biochemistry 302 February 24, 2006
Principles of gene regulation (cellular versus molecular level) Extracellular signals Environment (nutrient availability, temp.) Diffusible factors (hormones, growth factors, cytokines, chemokines) Less diffusible factors (cellcell, cell-matrix interactions) Intracellular effects Change in cell phenotype Specific changes in gene expression Seven processes affecting steady level/function of a protein Lehninger Principles of Biochemistry, 4th ed., Ch 28
Role of gene regulation in simple organisms vs metazoans Control of cell proliferation (growth and survival) Bacteria: environment Lower eukaryotes (yeast): environment Higher eukaryotes (mammals): constant environment Specific chemical signals Cell:cell/ECM interaction Control of cell differentiation (metazoans:development) Mediated by precise genetic & epigenetic 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 polymerase binding affinity Regulators of RNA polymerase isomerization Regulation of transcription factors by ligands To affect sequence-specific DNA-binding To affect protein-protein interaction Historical view (Jacob and Monod) KB k2 RNAP + P RNAP:Pc RNAP:Po affinity rate 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 to facilitate removal from cell) laci (or lacr): Lac repressor (regulates the laczya gene cluster) by binding to operator CRP site: camp receptor protein (mediates regulation based on glucose levels)
Early 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] (IPTG) lactose or allolactose in nature, IPTG used in lab setting Fig. 26-18 De-repression is necessary but not sufficient to activate the weak lac promoter.
Why the lac promoter is weak
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. Lac repressor can override CRP-induced activation.
Mapping of LacR binding sites in the lac regulatory region LacR and CRP DNAbinding sites (ciselements/operators) are slightly imperfect 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 One of the first regulatory proteins shown to bind DNA in a sequence-specific fashion Purified on the basis of IPTG affinity (Gilbert and Muller-Hill, 1966) Tetramer of four identical subunits (monomer 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 functionally relevant operator elements w/ Lac repressor 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 +11 82 93 bp loop Modularity of LacR tertiary structure with protruding DNAbinding domain Hinge N-terminal subdomain of core C-terminal subdomain of core Tetramerization helix: joins two dimeric units 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
Inducer (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)