Protein synthesis IIBiochemistry 302Two idealized views of the 70S ribosomal complex during translationProkaryotic translation: Cyclic nature of chain elongationRegeneration of GTP-EF-Tu by EF-Ts assisted nucleotide exchangePeptidyl transfer and translocation likely involves hybrid ribosome states (an idea championed by Harry Noller)A look at the transition state of peptidyl transferasePeptidyl transferase region of Haloarcula marismortuiBut what gives the RNA its catalytic power….making A2486 a stronger base via charge relayTermination of protein synthesisCounting the energy cost of translation (for a polypeptide of N residues)Translational efficiency enhanced by polyribosomes (elongation is rate-limiting)Summary of important differences in translation machinery in eukaryotesTranslation initiation in eukaryotesTranslational control in eukaryotesTranslational control by eIF2 kinases (regulation of globin synthesis in rbc)Many antibiotics are prokaryotic protein synthesis inhibitorsAntibiotics that bock protein synthesisMutations conferring antibiotic resistance map to 23S rRNA peptidyltransferase loopProtein synthesis IIBiochemistry 302Bob Kelm February 25, 2004Two idealized views of the 70S ribosomal complex during translation50S “tunnel”70S cavityFig. 27.25These models show all three sites (A, P, E) occupied by tRNAs. This would never occur during protein synthesis.View with 30S subunit in front, 50S subunit behindProkaryotic translation: Cyclic nature 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 and reloading of “free” EF-Tu with GTP via EF-Ts exchange factor• A,P sites (transpeptidation)– 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 hybrid-site– P-site tRNA (w/o peptide) - leaving group• A, P, E site (translocation, EF-G-GTP hydrolysis)– Transfer of uncharged tRNA to E site and ejection – Translocation of peptidyl-(3′OH) tRNA from A site to P site– Ribosome movement 3′ to the next codonModel for peptide chain elongation in prokaryotes30S subunit (Proofreading occurs after the charged tRNA is in place and both before and after GTP hydrolysis by EF-Tu.)50S subunit (Peptidyltransferase ribozymecomplex)EF-Tu-GTP is regenerated for another cycle.Fig. 27.22Regeneration of GTP-EF-Tu by EF-Ts assisted nucleotide exchangeNote that this exchange rxndoes not require GTP hydrolysis.This species is now ready to bind AA-tRNA for another round.Fig. 27.23after release from ribosomeready for another AA-tRNAPeptidyl transfer and translocation likely involves hybrid ribosome states (an idea championed by Harry Noller)EF-Tu: GDPProofreading3-nt stepChemistry can happen here.Anti-codon ends remain fixed in 30S subunit while acceptor ends of tRNAs are free to move leftward in 50S subunit.A look at the transition state of peptidyl transferase Tetrahedral carbon intermediate resolves to yield a deacylatedtRNA (P) and a peptidyl tRNA extended by one amino acid. P site A siteα3′Peptidyl transferase inhibitors with P or A site ribosome binding sites.AdenosinePuromycin resembles 3′end of amino-acylated tRNA.P. Nissen et al. Science 289:920-929, 2000Peptidyl transferase region of Haloarcula marismortuiNo proteins near (∼18 angstroms) of active site. Catalytic activity depends entirely on RNA.Atoms belonging to 23S rRNA >95% conserved in all three kingdoms are red.P. Nissen et al. Science 289:920-929, 2000But what gives the RNA its catalytic power….making A2486 a stronger base via charge relay3Negative 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 of A2486 is 3 Å from phosphoramide oxygen and 4 Åfrom amide N.2P. Nissen et al. Science 289:920-929, 2000P. Nissen et al. Science 289:920-929, 2000Raising the pKa of A2486 makes the proximal α amino group of AA-tRNA a better nucleophile3N3 represented as standard tautomer but is thought to function as a general baseTetrahedral carbon intermediate stabilized by H-bonding between protonated N3 and oxyanion.Deacylation: Proton transfer from N3 to the peptidyl-tRNA 3′OH.Termination of protein synthesis• Signaled by arrival of stop codon in the A site • No corresponding stop tRNA so release factor complex (RF1, RF2, RF3) binds to ribosome instead. RF3 is a GTPase.• Peptidyltransferase transfers P-site peptide chain to a water molecule. Release of peptide chain and RFs is stimulated by RF3-mediated hydrolysis of GTP.• Unstable 70S ribosome dissociates assisted by IF1 and IF3.• 30S subunit stays attached to polycistronic messages.Fig. 27.26Counting the energy cost of translation (for a polypeptide of N residues)• 2N: ATPs required to charge tRNAs (each charging reaction ATP → AMP + PPi)• 1: GTP needed for initiation• N-1: GTPs required for N-1 peptide bonds during elongation mediated by EF-Tu-GTP• N-1: GTPs required for N-1 translocations by EF-G-GTP• 1: GTP required for termination• Sum: 4N high-energy phosphate molecules must be hydrolyzed to complete a peptide chain of N residues– ∼160 kJ/mol per peptide bond– Entropy price paid by the cell for making a specific peptide sequence in a rapid and accurate way (~20Npossibilities if process were random)Translational efficiency enhanced by polyribosomes (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/RNA 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– 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 correct start Met• Initiation factors (multiple eIFs)– Many more required (11 vs 3)– Some bind mRNA, others attach to ribosomal subunits• Elongation factors (eEFs)– No differences, all orthologs of prokaryotic EFs• Termination (only one release factor)– eRF recognizes all stop codons (UAA, UAG, UGA)Translation