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Protein synthesis IIBiochemistry 302February 22, 2006Several idealized views of the 70S ribosomal complex during translationView with 30S subunit in front, 50S subunit behindBoth models imagine all three binding sites (A, P, E) occupied by tRNAs. This would only be a transientoccurrence during actual protein synthesis.50S “tunnel”70S cavityFig. 27.25Prokaryotic translation: Three steps of chain elongation• A site (AA-tRNA binding, EF-Tu-GTP hydrolysis)– Loading of new AA-tRNA joined to EF-Tu-GTP– Codon positioning of AA-tRNA assisted by GTP hydrolysis– Dissociation of EF-Tu-GDP (Released EF-Tu “reloaded” with GTP via EF-Ts exchange factor)• A,P sites (transpeptidation in 50S subunit)– α-amino group from A site AA-tRNA attacks the carbonyl carbon of P-site bound peptidyl-tRNA– Formation of new peptide bond at A/P 50S hybrid-site– P-site deacylated tRNA (i.e. w/o peptide) - leaving group• A, P, E site (translocation, EF-G-GTP hydrolysis)– Rapid transfer of uncharged tRNA to E site and ejection – Translocation of peptidyl-(3′OH) tRNA from A site to P site via EF-G-mediated ribosome movement 3′ to the next codonStage 3: Elongation Step 1: Binding of second aminoacyl-tRNA (EF-Tu-GTP)Regeneration of EF-Tu-GTP: This “reloading”cycle does not involve any GTP hydrolysis. Regeneration occurs by EF-Ts mediated nucleotideexchange.Proofreading of codon anticodon interaction likely occurs at this step to allow “incorrect” aminoacyl-tRNAsto dissociate. Lehninger Principles of Biochemistry, 4th ed., Ch 27GTPγS improves fidelity but reduces rate of synthesis.Stage 3: Elongation Step 2: Formation of first peptide bond (“mobile” tRNAs)Large 50S subunit: Peptidyltransferase ribozymecomplexSmall 30S subunit: Proofreading occurs after the charged tRNA is in place and both before and after GTP hydrolysis by EF-Tu.Structural constraints necessitate that both tRNAs likely shift their position in the 50S subunit to assume a hybrid binding states. There is no E site tRNA anticodon binding domain in 30S subunit.Lehninger Principles of Biochemistry, 4th ed., Ch 27αPeptidyl transfer and translocation likely involves hybrid ribosome states (an idea championed by Harry Noller)EF-Tu: GDPProofreading (ms time scale)3-nt stepChemistry can happen here.Stage 3: Elongation Step 3: Ribosome translocation to next codonEF-Tu + tRNA EF-G + GDP …ready for next cycle of elongation. Note how functional connection between mRNA template and the decoded growing polypeptide product is maintained.Facilitates change in ribosome conformation(A-site binding complex) (EF-G-GTP: a translocase)A look at the putative transition state of peptidyl transferase P. Nissen et al. Science 289:920-929, 2000Tetrahedral carbon intermediate resolves to yield a deacylatedtRNA (P) and a peptidyl tRNA extended by one amino acid. P site A siteAdenosineα3′Puromycin (an aminonucleosidedrug/antibiotic) resembles 3′ end of aminoacylatedtRNA.Peptidyl transferase inhibitors with P or A site ribosome binding sites.*Acceptor stem of tRNATyr: A-site analogHow puromycin inhibits protein synthesisStructure of peptidyl-puromycinPuromycin is made by the mold Streptomyces alboniger and affects both prokaryotic and eukaryotic ribosomes.Ribosome-selective antibiotic inhibitors• Cycloheximide– Affects 80S ribosome only – Blocks peptidyl transfer• Streptomycin– Bacterial 30S subunit specific– Causes codon misreading• Tetracycline– Bacterial A site drug – Block AA-tRNA binding (can’t pass thru euk cell membranes)• Chloramphenicol – Affects bacterial, mitochondrial, chloroplast ribosomes only– Blocks peptidyl transfer• Erythromycin– Binds 23rRNA of 50S subunit– Blocks elongation/translocation(mimics peptide bond)Atomic view of peptidyl transferase region of Haloarcula marismortuiP. Nissen et al. Science 289:920-929, 2000No proteins near (∼18 angstroms) of active site. Catalytic activity depends entirely on RNA.Atoms belonging to domain V of 23S rRNA >95% conserved in all three kingdoms are red.Catalytic potential achieved by making A2486 a stronger base via charge relayP. Nissen et al. Science 289:920-929, 20003Negative electrostatic charge originating from buried A2485 phosphate could be relayed to N3 of A2486 via the proposed mechanism to generate an imino tautomer.Charge relay mechanism is important in serine protease catalysis.imino N3 (pKa~1.5) of A2486 is ~3 Åfrom phosphor-amide oxygen and 4 Å from amide N.2N3 pKa~6.5P. Nissen et al. Science 289:920-929, 2000Raising the pKa of A2486 makes the proximal α amino group of AA-tRNA a better nucleophileTetrahedral carbon intermediate stabilized by H-bonding between protonated N3 imine group and oxyanion.N3 represented as standard tautomer but is thought to function as a general base.3Deacylation: Proton transfer from N3 to the peptidyl-tRNA 3′OH.33A siteP siteStage 4:Termination of polypeptide synthesis• Signaled by ribosome encountering a stop codon in the A site • No corresponding stop tRNA (except for Sec-tRNASec) so release factor complex binds to ribosome instead. – RF-1 (UAG, UAA), tRNA mimic– RF-2 (UGA, UAA), tRNA mimic– RF-3 (GTPase, needed to release 50S ribosome subunit)• Peptidyltransferase transfers P-site peptide chain to a water molecule. • Unstable 70S ribosome dissociates assisted by IF-1 and IF-3 perhaps.• 30S subunit likely stays attached to polycistronic messages and “slides”to next Shine-Dalgarno sequence.Lehninger Principles of Biochemistry, 4th ed., Ch 27Mechanisms ↑translational efficiency:couple transcription & translationThis figure is not drawn to scale →RNA polymerase (Mr~3.9 ×105) ribosome (Mr~2.7 × 106) and there are also many more proteins than mRNA molecules in a typical cell.Lehninger Principles of Biochemistry, 4th ed., Ch 27Translational efficiency enhanced by polysomes too (elongation is rate-limiting)One ribosome, one mRNA model does not account for the total rate of protein synthesis per E. coli cell.As many as 50 ribosomes bound per mRNA under certain conditions.Ribosome recyclingIn E. coli, 15,000 ribosomes synthesizing @ 15 AA/sec →750 proteins of 300 AA/sec.Fig. 27.29Summary of important differences in translation machinery in eukaryotes• Ribosome (polysomes and attached to ER)– Additional 5.8S rRNA component in large 60S subunit – mRNA aligned on the small 40S subunit using 5′ cap (no Shine-Dalgarno sequence or fMet)– Scanning identifies


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UVM BIOC 302 - Protein synthesis II

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