Protein Synthesis II Elongation Termination Post Transcriptional Regulation Lecture 7 ELONGATION 4 steps 1 2 3 4 AA ac va on see previous lecture on ini a on Aminoacyl tRNA aa tRNA binds A site eEF 1 Elonga on Factor 1 escorts aa tRNA to A site eEF 1 G protein Ribosome accepts aa tRNAs 2 sites A site Aminoacyl tRNA site P site Pep dyl tRNA site A site is only lled by aa tRNA if P site has a pep dyl tRNA AA ac va on aa tRNA binds A site eEF 1 takes aa tRNA to A site ribosome accepts Protein Synthesis Elonga on eEF 1A Euk Elonga on Factor 1A eEF 1A GTP is ac ve high a nity for ribosome aa tRNA base pairing b tw tRNA mRNA codon triggers GTPase GAP hydrolysis ribosome or some elonga on factor has a GAP ac vity eEF 1A GDP must be released from ribosome before a pep de bond forms eEF 1A GTP must be regenerated to SUSTAIN transla on eEF 1 Euk Elonga on Factor 1 eEF 1 GNEF for eEF 1A eEF 1A eEF 1 eEF 1A eEF 1A GTP ac ve w a nity for ribosome aa tRNA bp ing b tw tRNA mRNA codon triggers GTPase GAP ac vity eEF 1A GDP releases so pep de bond can form eEF 1A GTP regenerate to sustain transla on eEF 1 GNEF for eEF 1A eEF 1A GTP ribosome aa tRNA eEF 1 eEF 1A GDP pep de bond eEF 1A GTP Protein Synthesis E T eEF 1 PEPTIDE BOND Forma on nitrogen very ELECTRONEG amino group a acks ester makes amide pep de bond 1 aa tRNA grows by nx AAs 2 Pep dyl tRNA is deacylated 3 Protein length increases from C terminus end 4 Amino group of aa tRNA a acks ester on pep dyl tRNA 5 Ester breaks amide forms energe cally favorable 2 esters a ached to AA in A site aa tRNA grows p tRNA deacylates length inc amino on aa tRNA a acks ester on p tRNA Protein Synthesis E T Pep de Bond PEPTIDYL TRANSFERASE General Acid Base Catalysis by 28S rRNA adenine from 28S rRNA a ribozyme free tRNA new tRNA aFached to nascent protein charged tRNA in A site a acks nascent protein in P site Protein Synthesis E T Pep dyl Transferase Reac on PEPTIDE BOND tRNA Arrangement ALer Forma on Transient Presence of Pep dyl tRNA A Site aHer pep de bond forma on pep dyl tRNA A site deacylated tRNA P site ribosome CANNOT con nue w transla on in this state new aa tRNA cannot enter A site solu on transloca on p tRNA in A site deacyl tRNA in P site no new aa tRNA enters Protein Synthesis E T tRNA Arrangement Post Amide Bond TRANSLOCATION Rela ve Movements of mRNA Ribosome 60S subunit moves P site recons tuted E site occupied by deacylated tRNA A site incomplete 40S subunit moves A site re formed new codon A site transloca on costs 1 GTP catalyzed by EF G in proks eIF 2 in euks GTP hydrolysis 60S P site re lled E site occupied A site empty 40S A site re formed re lled Protein Synthesis E T Transloca on eEF 2 Euk Elonga on Factor 2 eEF 2 Required for Transloca on translocase GTP binding protein GTP hydrolysis required for transloca on translocase GTP binding protein GTP hydrolysis required for transloca on translocase GTP binding protein need GTP hydrolysis for transloca on Protein Synthesis E T eEF 2 TERMINATION mRNAs have 1 of 3 termina on codons UAG amber UAA ochre UGA opal no matching tRNAs for termina on termina on codons recognized by release factors RFs A site RF binding to A site requires pep dyl tRNA P site RF gets chain to transfer to H2O 3 termina on codons recognized by RFs A site requires pep dyl tRNA P site Protein Synthesis Termina on TERMINATION Overview post termina on complex release factor binds A site protein cleaved from tRNA puromycin an bio c premature termina on nascent pep de chain transferred to puromycin instead of H2O ribosome recycling instead of breaking ester to form amide con nue chain add H2O over ester so essen ally adding nothing RF binds A site protein cleaved from tRNA H2O post term complex rib recycles Protein Synthesis Termina on Overview RELEASE FACTORS 1 euk RF eRF 1 works w eRF 3 sees term codon A site makes ribosome hydrolyze ester on pep dyl tRNA eRF 3 GDP controls dissocia on of eRF 1 euks have 1 RF eRF 1 Euk Release Factor 1 eRF 1 works together w eRF 3 recognizes termina on codon A site causes ribosome to hydrolyze ester bond on pep dyl tRNA dissocia on of eRF 1 controlled by eRF 3 GDP Hydrolysis of Pep dyl tRNA Ester on Ribosome pep dyl transferase center esterase eRF 1 works w eRF 3 sees term codon causes ester hydrolysis control eRF 3 GDP Protein Synthesis E T Euk Release Factors GTP binding protein GTPase pep de release occurs before eRF 3 GTP eRF 3 binds eRF 1 eRF 3 GTP binds eRF 1 eRF 1 GAP for eRF 3 eRF 3 GDP causes release of eRF 1 from ribosome eRF 3 GDP dissociates from ribosome GTP binding protein pep de release eRF 3 GTP GAP for eRF 1 binds eRF 1 eRF 3 GDP causes eRF 1 to release eRF 3 GDP dissociates GTP binding pep de release eRF 3 GTP binds eRF 1 eRF 1 releases eRF 3 GDP dissociates Protein Synthesis E T eRF 3 eIF 1A eIF 3 coopera vely dissociate RIBOSOME RECYCLING eIF 1 causes dissocia on of tRNA eIF 3J facilitates dissocia on of mRNA 60S from 40S from P site from 40S eIF 1A eIF 3 dissociate 60S from 40S eIF 1 dissociates tRNA from P site eIF 3J facilitates mRNA dissocia on from 40S eIF 1A eIF 3 60S from 40S eIF 1 tRNA from P site eIF 3J mRNA from 40S Protein Synthesis E T Removal of Discharged tRNA POST TRANSCRIPTIONAL REGULATION protein mediated regula on RNA mediated control Mechanisms Post Transcrip onal Modi ca ons of mRNAs block ini a on of protein synthesis eIF 4 had to recognize cap start scanning bring it all together prevent use of mRNA for protein synthesis PERK eIF 2 TOR eIF 4 iron shorten mRNA half life microRNA block ini a on prevent use of mRNA or shorten mRNA half life Protein Synthesis Regula on Post Transcrip onal Reg POST TRANSCRIPTIONAL REG eIF 2 Ini a on 1 Phosphoryla on of eIF 2 by eIF 2B Regulates Protein Synthesis Ini a on 4 kinases can regulate eIF 2 GCN2 General Control Non Repressed PKR Protein Kinase R HRI Heme Regulated Inhibitor Kinase PERK Protein Kinase ER Localized R like eIF 2 Kinase 2 3 4 too many unfolded proteins in ER not enough proteins all 4 kinases phosphorylate eIF 2 if you e up eIF 2B then there s no more eIF 2B deprive cell of its GAP can t have any more …
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