Lecture 25 Chapters 39 40 Translation peptides mRNA ribosomes http bioap wikispaces com Ch 17 Collaboration 1 Outline Ribosomal subunits Initiation complex formation Elongation Termination Prokaryotic vs eukaryotic protein synthesis Synthesis of secreted proteins Regulation of protein synthesis Mutations 2 The Prokaryotic Ribosome 23S RNA 16S RNA 5S RNA 34 L protein 60S 21 S protein In eukaryotes 80S 40S 3 The ribosome has three binding sites for tRNAs Peptidyl transferase Center A aminoacyl tRNA site P peptidyl tRNA site E exiting tRNA site Decoding Center 4 Initiation in Prokaryotes 1 Ribosome Binding Site on mRNA sometimes called a Shine Dalgarno sequence base pairs with 16S rRNA of 30S subunit 2 Initiation codon AUG 3 Initiation factors IF IF 1 IF 2 GTP IF 3 4 Initiator tRNA fMet tRNAiMet 5 Initiation Site purine rich Shine Dalgarno Sequences Ribosome Binding Site UAC 6 E coli Initiation Factors IF1 Binds to 30S A site and prevents tRNA binding IF2 G protein that binds fMet tRNA initiator tRNA which docks at P site of 30S IF3 Binds to 30S E site prevents 50S binding 7 Table 30 7 p 967 Initiator tRNA 8 Formation of the 30S initiation complex IF3 RBS 9 Formation of the 70S initiation complex 50S E P A mRNA 30S 10 E coli Elongation Factors EF EF Tu G protein that binds aminoacyl tRNA and delivers it to the A site EF Ts Guanine nucleotide exchange factor GEF that replaces GDP on EF Tu with GTP EF G G protein that promotes translocation of mRNA 11 EF Tu brings charged tRNA to appropriate codon 12 EF Tu brings charged tRNA to appropriate codon EF Tu GTP P GT Charged tRNA 70S initiation complex EF Tu GDP P GD 13 Resetting EF Tu by EF Ts EF Ts Is the GTP Exchange Factor EF Tu GTP GTP EF Tu GTP P GT GTP Charged tRNA EF T EF Tu GDP P GD To ribosome Charged tRNA P GD 14 Covalent linkage of amino acid to cognate tRNA 3 3 2 5 15 Peptide bond formation 16 Polypeptide Chain C N NH3 a a 1 COOa a 2 a a 3 a a 4 a a 5 17 Peptide Bond Synthesis is Catalyzed by the 23S RNA not by a protein enzyme the Ribosome is a Ribozyme peptidyl trnasferase The 50S subunit 3 end of P site tRNA is red 3 end of A site tRNA is green There is no protein near only RNA 18 Peptide bond formation by peptidyl transferase P 50S Peptidyl transferase Center E E N P 1 2 3 30S codons Decoding Center E P 19 Translocation by EF G E P E P E P E P 20 EF G Mimics EF Tu tRNA Protein EF Tu tRNA EF G 21 An Active Ribosome A aminoacyl P peptidyl E exit Peptidyl transferase Center Decoding Center 22 Polypeptide chain growth N N N C tRNA1 tRNA2 23 Translation Elongation fM et a a 5 aa a a aa aa 50S CG A 50S aa UU C UAG CCC AAC GCU AUC GGG UUG AAG CUC E 30S P A Ribosome Movement 24 Translation Elongation fM et a a 50S aa a aaa aa aa U A G UU C 5 CCC AAC GCU AUC GGG UUG AAG CUC E 30S P A Ribosome Movement 25 Translation Elongation fM et a a aa a a 50S aa aa aa UU C 5 CCC AAC GCU AUC GGG UUG AAG CUC E 30S P A Ribosome Movement 26 Translation Elongation fM et a a aa a a 50S aa aa aa aa GA G 5 CCC AAC UUC GCU AUC GGG UUG AAG CUC E 30S P A Ribosome Movement 27 Translation Elongation a fMetaa aa a a aa a 50S aa aa CC C GA G 5 AAC UUC GCU AUC GGG UUG AAG CUC E 30S P A Ribosome Movement 28 Translation Elongation fM et a a aa a aaa 50S aa aa aa GA G C C C 5 AAC UUC GCU AUC GGG UUG AAG CUC E 30S P A Ribosome Movement 29 Translation Elongation fM et a a 5 aa a aaa 50S aa aa aa AAC UUC GAG GCU AUC GGG UUG AAG CUC E 30S P A Ribosome Movement 30 fM aa et aa aa 50S aa aa aa a a C A A 5 UUC GAG GCU AUC GGG UUG AAG CUC E 30S P A Ribosome Movement 31 fM et a a 5 aa 50S aaa a a a a a aa UUC GAG GCU AUC GGG UUG AAG CUC AUG E 30S P A Ribosome Movement 32 Termination of protein synthesis in prokaryotes As the ribosome is translocating along the mRNA 5 3 elongation terminates when the ribosome decoding center encounters a STOP codon UAA UAG UGA on the mRNA RF 1 recognizes the STOP codons UAA and UAG RF 2 recognizes the STOP codons UAA and UGA RF 3 GTPase that removes RF 1 and RF 2 from the ribosome RRF ribosome release factor RF 2 Protein tRNA tRNA EF Tu 33 Complex Translation Termination 50S 1 or 2 Peptidyl tRNA link hydrolyzed 30S 34 When RF 3 GDP is converted to RF 3 GTP RF 1 dissociates 35 Hydrolysis of GTP to GDP causes RF 3 to dissociate from the ribosome 36 When translation terminates ribosomes dissociate into subunits that are recycled RRF ribosome recycyling factor 37 RRF is another example of molecular mimicry it looks like a tRNA 38 EF G GTP binds to the A site and when the GTP is hydrolyzed to GDP translocates RRF to the P site 39 This causes the whole complex to dissociate and IF 3 then binds to the E site to prevent the 50S subunit from rebinding 40 Energy Requirements for Translation 1 ATP to charge a tRNA plus 1 PPi 2 ATP equivalent 1 GTP for EF Tu to deliver the aminoacyl tRNA to the ribosome 1 GTP for EF G to translocate the ribosome GTP ATP for energy Therefore 4 ATP equivalent per amino acid added or 4 high energy phosphoanhydride bonds Also use one IF 2 GTP per initiation and two GTP RF 3 and EF G use one each per termination 41 Eukaryotes Translation differs from Prokaryotes Bigger ribosomal subunits Met tRNA is the initiator aminoacyl t RNA not formyl Met tRNA Eukaryotic mRNA has NO Ribosome Binding Site the cap is recognized instead Structure of mRNA Many more initiation elongation and termination factors Organization 42 Prokaryotic mRNA DNA RBS Polycistronic mRNA More than one coding region Protein B Protein c Protein A Ribosomes translating mRNA into proteins A B AND C 43 Translation in Eukaryotes Initiation codon 44 Eukaryotic translation Initiation eIF 40S 45 Formation of the 80S complex 60S 40S 46 Protein interactions circularize eukaryotic mRNA 47 Synthesis of secretory and membrane proteins Rough reticulum 48 49 50 51 Regulation of protein synthesis Iron response element binding protein X of ferritin 52 Regulation of protein synthesis Fe of ferritin Ferritin Fe 53 Regulation of protein synthesis Transferrin receptor mRNA Transferrin receptor protein 54 Regulation of protein synthesis Transferrin receptor mRNA …
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