Protein Synthesis I Initiation Lecture 6 Features of Euk mRNAs 5 end 5 cap bound by protein synthesis ini2a2on factors how we know mRNA is ready for transla2on 5 UTR Untranslated Region contributes to selec2on of transla2on start site 3 end poly A tail in uences ini2a2on of transla2on 5 cap bound by IFs 5 UTR start site selec2on poly A tail ini2a2on of transla2on Protein Synthesis Ini2a2on mRNA RIBOSOME ribosome transla2on machine 4 ribosomal RNAs rRNAs 82 polypep2des mRNA template for protein synthesis transla2on machine 4 rRNAs mRNA template Protein Synthesis Ini2a2on Ribosome Protein Synthesis Road Map 1 2 3 4 5 6 7 AAs aQached to tRNAs aminoacylG tRNA synthetases Delivery of aminoacylTtRNAs to ribosome iniHaHon factors Ribosome nds mRNA UTRs iniHaHon factors Selec2on of correct ini2a2on codon reading frame tRNA ribosome Pep2de bond forms ribosome Elonga2on of pep2de chain ribosome Termina2on of polypep2de synthesis release factors ribosome tRNAs delivery to ribosome mRNA ini2a2on codon pep2de bond elonga2on termina2on Protein Synthesis Road Map joins AA to its tRNA each AA has a speci c aminoacylGtRNA synthetase can charge several tRNAs w di erent an2codons tRNA charging occurs 3 terminus an invariant A of tRNA AA links to invariant A via an ester bond to 2 or 3 OH AMINOACYL GtRNA SYNTHETASE joins AA to tRNA speci c aminoacylGtRNA synthetase for each AA charge several tRNAs w di erent an2codons occurs 3 terminus AA links to invariant A via ester bond to 2 or 3 OH aminoacyl AMP intermediate formed transferred to tRNA all end w same sequence CCA this is where AA goes joins AA to tRNA speci c synthetase for each AA tRNA charging ester bond Protein Synthesis Ini2a2on AminoacylTrRNA Synthetase AMINOACYLGtRNA SYNTHETASE REACTION tRNA Charging ReacHon AMP beQer leaving group than a hydroxyl TOH acHvaHon cut 2 PO4 s o of ATP break bond to make ester make this highTenergy compound to drive reac2on aQach PO4 5 end charging of tRNA ester will break ester bond in ribosome to make amide pepHde bond which is more stable ac2va2on reac2on tRNA charging reac2on ester forms Protein Synthesis Ini2a2on Charging Reac2on AMINOACYL tRNA SYNTHETASE Proofreading preTtransfer edi2ng reject as aaTAMP i e AA on AMP postTtransfer edi2ng reject as aaTtRNA i e afer AA is added to tRNA edi2ng likely catalyzed by tRNA making it an ancient ribozyme a ribosome CANNOT reject a wronglyTcharged tRNA so don t want to have incorrect tRNA hence the edi2ng blah Protein Synthesis Ini2a2on Proofreading tRNA As an Adaptor an2codon of tRNA ensures selec2on of correct aminoacylTtRNA by ribosome 5 3 UUC can baseTpair w this an2codon an2codon makes sure correct aminoacylTtRNA is selected by ribosome Protein Synthesis Ini2a2on tRNA as an Adaptor INITIATION General Aspects proteins synthesized from amino N T terminus methionine ini ator AA mRNA is read in triplets gene2c code 5 UTR sequence NOT part of protein a mRNA has 3 reading frames 3 possible proteins reading frame selected ini ator Met eIF Eukaryo2c Ini2a2on Factor brings ini ator AA methionine to correct place MetGtRNAi charged ini ator tRNAMet di erent from regular tRNAm proteins made from NTterminus Met ini2ator AA 3 RFs selected by Met eIF Protein Synthesis Ini2a2on General Aspects TERNARY COMPLEX contains MetGtRNA eIFG2 GTP binds 40S eIFG3 eIFG1A eIFG1 43S complex eIFG2 eIF3 contains MetGtRNA eIFG2 GTP binds 40S eIFG3 eIFG1A eIFG1 eIFG2 GTPTbinding protein eIFG3 stabilizes binding of ternary complex to 40S ribosomal subunit 43S complex MetTtRNA eIFT2 GTP 40S eIFT3 eIFT1A eIFT1 43S complex eIFT2 eIFT3 Protein Synthesis Ini2a2on Preparing Ini2ator MetTtRNA eIFG4 Prep Recruitment of mRNA Template eIFG4F contains 4A 4E 4G binds 5 cap of mRNA 4A helicase unwinds opens mRNA so it can be scanned 4E binds 5 cap 4G recognizes poly A Tbinding protein PABP is bound to 5 cap eIFG4A eIFG4B helicases unwind duplex regions of 5 UTR eIFG4G RNA unwinding by eIFG4A requires ATP hydrolysis secondary structure in RNA prevents ribosome movement along mRNA obscures important singleTstranded sites e g AUG binds mul2ple proteins mRNA eIFG4E eIFG4A eIFG3 eIFG1 mRNA PABP helps 43S complex bind mRNA binds eIFG3 mRNA coordinates ini2a2on of protein synthesis eIFT4F 4A 4E 4G eIFT4A eIFT4B eIFT4G Protein Synthesis Ini2a2on Where is the mRNA CAP BINDING COMPLEX CBC FuncHon as Central Player for eIFG4G 43S ribosome binds CBC CapTBinding Complex CBC eIFG4F poly A tail 43S iniHaHon complex eIFG3 RNATbinding proteins RNA structure ini2a2on codon ds must be unwound so iniHaHon complex 43S can scan to start site thus scanning by 43S complex requires helicases poly A tail 43S ini2a2on complex ini2a2on codon Protein Synthesis Ini2a2on ProteinTProtein Interac2ons on mRNA 3 5 SelecHon of IniHaHon Site The Linear Scanning Model iniHaHon complex scans mRNA to locate aug in a good context T3 to T1 consensus of vertebrate ini2a2on codons A GNCaug N any nucleo2de movement of 43S ribosome to aug requires eIFG1A eIFG1 Linear Scanning Model ini2a2on complex scans mRNA Protein Synthesis Ini2a2on Movement of 43S Complex to Ini2a2on Codon AMINOACYLGtRNAs On Ribosomes both ribosome subunits are needed to form AGsite E Exi2ngTtRNA P Pep2dylTtRNA start codon only AA that enters PTsite all other AAs codons enter ATsite A AminoacylTtRNA ATsite forma2on requires both ribosome subunits Protein Synthesis Ini2a2on tRNA Binding Sites on Ribosome 60S RIBOSOME Docking on 48S Complex Docking of 60S Ribosome on 48S Complex requires eIFG5 eIFG5B eIFG5 GAP for eIFG2 when eIFG2 is on ribosome eIFT5 checks to see if eIFT2 is there eIFT5 does GAP ac2vity on eIFT2 eIFT2 hydrolyzes falls o dissociates causes dissocia2on of eIFG2 eIFG3 from ribosome eIFG5B GTP binds ribosome a er release of eIFG2 GTP 60S ribosome arrival binds a er eIFG5B associates w 48S complex 60S ribosome causes dissocia2on of eIFG5B eIFG1 eIFG3 eIFG1A from 48S complex MetGtRNA is PTsite requires eIFT5 eIFT5B 60S ribosome arrival Protein Synthesis Ini2a2on 60S Ribosome Arrival Forma2on of PTSite AG PGSITES FuncHons During IniHaHon importance of PTsite occupancy to allow more AAs to enter ATsite rRNA catalyzes forma2on of pep2de bond translocaHon importance of P site occupancy to allow AA to enter ATsite Protein Synthesis Ini2a2on Importance of PTSite Occupancy eIFG2B eIFG2 released as eIFG2 GDP eIFG2B catalyzes GDP GTP exchange GAP for eIFT2 regenerates ac ve eIFG2 GTP eIFG2 released as eIFG2 GDP eIFG2B catalyzes GDP GTP exchange regenerates ac2ve eIFG2 GTP eIFG2B