Lecture 25 Chapters 39 40 Transla7on pep7des mRNA ribosomes h9p bioap wikispaces com Ch 17 Collabora7on 1 Outline Ribosomal subunits Ini7a7on complex forma7on Elonga7on Termina7on Prokaryo7c vs eukaryo7c protein synthesis Synthesis of secreted proteins Regula7on of protein synthesis Muta7ons 2 The Prokaryo7c 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 Pep7dyl transferase Center A aminoacyl tRNA site P peptidyl tRNA site E exiting tRNA site Decoding Center 4 Ini7a7on 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 Ini7a7on Site purine rich Shine Dalgarno Sequences Ribosome Binding Site UAC 6 E coli Ini7a7on Factors IF1 Binds to 30S A site and prevents tRNA binding IF2 G protein that binds fMet tRNA ini7ator tRNA which docks at P site of 30S IF3 Binds to 30S E site prevents 50S binding 7 Table 30 7 p 967 Ini7ator tRNA 8 Forma7on of the 30S ini7a7on complex IF3 RBS 9 Forma7on of the 70S ini7a7on complex 50S P E 30S A mRNA 10 E coli Elonga7on Factors EF EF Tu G protein that binds aminoacyl tRNA and delivers it to the A site EF Ts Guanine nucleo7de exchange factor GEF that replaces GDP on EF Tu with GTP EF G G protein that promotes transloca7on of mRNA 11 EF Tu brings charged tRNA to appropriate codon 12 EF Tu brings charged tRNA to appropriate codon EF Tu GTP Charged tRNA 70S ini7a7on complex EF Tu GDP 13 Resetting EF Tu by EF Ts EF Ts Is the GTP Exchange Factor EF Tu GTP EF Tu GTP Charged tRNA EF Tu GDP EF T To ribosome Charged tRNA 14 Covalent linkage of amino acid to cognate tRNA 3 3 2 5 15 Pep7de bond forma7on 16 Polypep7de Chain N C NH3 a a 1 COO a 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 Pep7de bond forma7on by pep7dyl transferase P 50S Pep7dyl transferase Center E E P 1 2 3 30S codons Decoding Center E P 19 Transloca7on 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 Ac7ve Ribosome A aminoacyl P pep7dyl E exit Pep7dyl transferase Center Decoding Center 22 Polypep7de chain growth N N N C tRNA1 tRNA2 23 Translation Elongation 50S aa aa 50S 5 UAG CCC AAC GCU AUC GGG UUG AAG CUC E P 30S A Ribosome Movement 24 Translation Elongation 50S 5 CCC AAC GCU AUC GGG UUG AAG CUC E P 30S A Ribosome Movement 25 Translation Elongation 50S aa 5 CCC AAC GCU AUC GGG UUG AAG CUC E 30S P A Ribosome Movement 26 Translation Elongation 50S aa 5 aa CCC AAC UUC GCU AUC GGG UUG AAG CUC E 30S P A Ribosome Movement 27 Translation Elongation fMet aa aa aa aa aa 50S 5 AAC UUC GCU AUC GGG UUG AAG CUC E 30S P A Ribosome Movement 28 Translation Elongation 50S aa 5 AAC UUC GCU AUC GGG UUG AAG CUC E 30S P A Ribosome Movement 29 Translation Elongation 50S aa 5 aa AAC UUC GAG GCU AUC GGG UUG AAG CUC E P 30S A Ribosome Movement 30 50S 5 UUC GAG GCU AUC GGG UUG AAG CUC E 30S P A Ribosome Movement 31 50S aa 5 UUC GAG GCU AUC GGG UUG AAG CUC AUG E P 30S A Ribosome Movement 32 Termina7on of protein synthesis in prokaryotes As the ribosome is transloca7ng along the mRNA 5 3 elonga7on 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 Pep7dyl 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 ini7a7on and two GTP RF 3 and EF G use one each per termina7on 41 Eukaryotes Transla7on di ers from Prokaryotes Bigger ribosomal subunits Met tRNA is the ini7ator aminoacyl t RNA not formyl Met tRNA Eukaryo7c mRNA has NO Ribosome Binding Site the cap is recognized instead Structure of mRNA Many more ini7a7on elonga7on and termina7on factors Organiza7on 42 Prokaryo7c mRNA DNA RBS Polycistronic mRNA More than one coding region Protein B Protein c Protein A Ribosomes transla7ng mRNA into proteins A B AND C 43 Transla7on in Eukaryotes Ini7a7on codon 44 Eukaryo7c transla7on Ini7a7on eIF 40S 45 Forma7on of the 80S complex 60S 40S 46 Protein interac7ons circularize eukaryo7c mRNA 47 Synthesis of secretory and membrane proteins Rough re7culum 48 49 50 51 Regula7on of protein synthesis Iron response element binding protein X of ferriDn 52 Regula7on of protein synthesis Fe of ferriDn Ferri7n Fe 53 Regula7on of protein synthesis Transferrin receptor mRNA Transferrin receptor protein 54 Regula7on of protein synthesis Transferrin receptor mRNA X Transferrin receptor Fe Fe 55 Mutation and Mutagenesis Some Terminology Mutant An organism that differs in DNA sequence from the wild type Mutation A stable heritable change in the DNA sequence Can be spontaneous or induced Mutagen A physical or chemical agent that induces mutations 56 Point Mutations Consequences for Proteins Silent Mutation GAG GAA Missense Mutation GAG AAG Nonsense Mutation Glu Glu Glu Lys GAG TAG Glu STOP Mutations are commonly designated by wild type amino acid position in the protein mutant amino acid For example E214K The mutation resulted in a Glu at amino acid position 214 changed to Lys 57 Insertion Deletion Mutations THE CAT AAT ETH EFA …
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