Initiation of translation in eukaryotic cells:connecting the head and tail1: 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 translation3: Identification of start AUG achieved by “scanning” mechanism involving eIF4B and eIF4F(complex of 4E, 4A, and 4G). Initiator tRNA is Met-tRNAMet.GCCRCCAUGGRecoding of internal UGA codon as Sec• Sec-tRNASec(specific tRNA)• eEFSecor 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/ eEFSec:GTP:Sec-tRNASec– can bind ribosomes and 28S rRNA• ribosomal protein L30 (rpL30)?– enhances incorporation of Sec in transfected cells– competes with SBP for interaction with SECISModels from S. A. Kinzy et al. Nucleic Acids Res. 33, 5172 (2005); M. Leibundgutet al. EMBO J. 24, 11 (2005)Gene regulation IBiochemistry 302February 24, 2006Principles of “gene regulation”(cellular versus molecular level)• Extracellular signals– Environment (nutrient availability, temp.)– Diffusible factors (hormones, growth factors, cytokines, chemokines)– Less diffusible factors (cell-cell, cell-matrix interactions)• Intracellular effects– Change in cell phenotype – Specific changes in gene expression– Seven processes affecting steady level/function of a proteinLehninger Principles of Biochemistry, 4th ed., Ch 28Role 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)messengerThe 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)RNAP + P RNAP:Pc RNAP:PoelongatingcomplexKkB2affinity rateSugar 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. colimutants 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 productLehninger Principles of Biochemistry, 4th ed., Ch 28Structure of the lactose (lac) operon (structural genes + regulatory elements)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 operatorCRP site: cAMP receptor protein (mediates regulation based on glucose levels)Fig. 26-17Early operon model based entirely on indirect evidence from genetic analysisDe-repression is necessary but not sufficient to activate the weak lac promoter.Primary control via repression!Fig. 26-18lactose or allolactose in nature, IPTG used in lab settingGal(β1→4)GlcGal(β1→6)Glchigh [glc]low [glc] high [lac](IPTG)Why the lac promoter is weaklac operon is co-regulated by changes in Lac repressor and CRP activator bindingTrans-activation not predicted by Jacob and Monod, also thought that the Lac repressor was RNA.Fig. 26-21Isopropyl β-thiogalactoside (IPTG) used in the labcAMP receptor protein (CRP) or catabolite activator protein (CAP)CRP-cAMP (inducer) complex binding to target site occurs when glucose is absent or very low.Lac repressor can override CRP-induced activation.Mapping of LacRbinding sites in the lac regulatory regionLacR and CRP DNA-binding sites (cis-elements/operators) are slightly imperfect inverted repeats.Fig. 26-19Binding affinity: O1 (main operator) >O2or O3(secondary operators)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 (∼10-8M)• Binds specifically to operator (Kd= ~10-10M) and non-specifically to DNA (Kd= ~10-5M).– In response to inducer binding, repressor affinity changes from ‘specific’ to ‘non-specific’ mode.– Three binding sites centered at +11 (O1), −82 (O3), and +432 (O2) 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+11 −82• Tetramerization helix: joins two dimeric units• Core domain: binds inducers e.g. IPTG, allolactose• Headpiece: DNA-binding domain comprised of hinge & helix-turn-helix motif93 bp loopLewis, M. et al. (1996) Science 271:1247HTHHingeN-terminal subdomainof coreC-terminal subdomainof coreT HelixModularity of LacR tertiary structure with protruding DNA-binding domainMolecular 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 28Inducer (IPTG) may serve to drive Lac repressor DNA-binding helices apartLewis, M. et al. (1996) Science 271:1247no