Lecture'25'*'Chapters'39*40'Transla7on'h9p://bioap.wikispaces.com/Ch+17+Collabora7on'ribosomes'pep7des'mRNA'1'Outline'• Ribosomal'subunits'• Ini7a7on'complex'forma7on'• Elonga7on'• Termina7on'• Prokaryo7c'vs.'eukaryo7c'protein'synthesis'• Synthesis'of'secreted'proteins'• Regula7on'of'protein'synthesis'• Muta7ons'2'23S'RNA'5S'RNA'16S'RNA'21'S'protein'34'L'protein'The'Prokaryo7c'Ribosome'(60S)' (80S)' (40S)'In'eukaryotes'3'The ribosome has three binding sites for tRNAs A = aminoacyl-tRNA site P = peptidyl-tRNA site E = exiting tRNA site Decoding'Center'Pep7dyl'transferase'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'Table'30*7,'p.'967'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'Ini7ator'tRNA'8'Forma7on'of'the'30S'ini7a7on'complex'IF3)RBS'9'Forma7on'of'the'70S'ini7a7on'complex'50S'30S'mRNA)P''''A'''E'''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'12'70S'ini7a7on'complex'EF*Tu'brings'charged'tRNA'to'appropriate'codon'EF*Tu*GTP'EF*Tu*GDP'Charged)tRNA)13'EF*Tu*GTP'EF*Tu*GDP'Charged)tRNA)Charged)tRNA)To'ribosome'EF*Tu*GTP'EF*T'Resetting EF-Tu by EF-Ts EF-Ts Is the GTP Exchange Factor 14'3’'2’'Covalent'linkage'of'amino'acid'to'cognate'tRNA'5’'3’'15'Pep7de'bond'forma7on'16'Polypep7de'Chain'N’ç'è'C’'a.a.'2'a.a.'3'a.a.'4'a.a.'5'NH3+))a.a.'1'COO.)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. 3ʼ end of P-site tRNA is red, 3ʼ end of A site tRNA is green. There is no protein near, only RNA. 18'Pep7de'bond'forma7on'by'pep7dyl'transferase'E)P)50S'30S'E)P)E)P)1)2) 3)codons'Decoding'Center'Pep7dyl'transferase'Center'19'Transloca7on'by'EF*G'E)P)E) P)E)P)E)P)20'EF-G Mimics EF-Tu-tRNA EF-Tu-tRNA EF-G Protein 21'An'Ac7ve'Ribosome'A='aminoacyl'P'='pep7dyl'E='exit'Decoding'Center'Pep7dyl'transferase'Center'22''Polypep7de*chain'growth'N’'N’'N’'C’'tRNA1'tRNA2'23'5ʼ GCU AUC GGG UUG AAG CUC CCC aa AAC aa Ribosome Movement UAG E P A 50S'50S'Translation Elongation'30S'24'5ʼ GCU AUC GGG UUG AAG CUC CCC AAC Ribosome Movement E P A 50S'Translation Elongation'30S'25'E P A 5ʼ GCU AUC GGG UUG AAG CUC CCC AAC aa Ribosome Movement 50S'Translation Elongation'30S'26'E P A 5ʼ GCU AUC GGG UUG AAG CUC AAC aa Ribosome Movement UUC aa CCC Translation Elongation'30S'50S'27'Ribosome Movement E P A 5ʼ GCU AUC GGG UUG AAG CUC AAC fMet-aa-aa-aa-aa-aa- UUC Translation Elongation'30S'50S'28'Ribosome Movement E P A 5ʼ GCU AUC GGG UUG AAG CUC AAC UUC aa Translation Elongation'30S'50S'29'Ribosome Movement E P A 5ʼ GCU AUC GGG UUG AAG CUC GAG aa AAC UUC aa Translation Elongation'30S'50S'30'Ribosome Movement E P A 5ʼ GCU AUC GGG UUG AAG CUC GAG UUC 30S'50S'31'Ribosome Movement E P A 5ʼ GCU AUC GGG UUG AAG CUC AUG GAG aa UUC 30S'50S'32'As'the'ribosome'is'transloca7ng'along'the'mRNA'(5ʹ′'à'3ʹ′),'elonga7on'terminates'when'the'ribosome'decoding'center'encounters'a'STOP'codon'(UAA,'UAG,'UGA)'on'the'mRNA.'Termina7on'of'protein'synthesis'in'prokaryotes • 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 Complex 33'Translation Termination Pep7dyl*tRNA'link'hydrolyzed'(1'or'2)'50S'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'differs'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'Polycistronic'mRNA''(More'than'one'coding'region)'DNA'Protein'A'Protein'B'Protein'c'RBS'Ribosomes'transla7ng'mRNA'into'proteins'A,'B,'AND'C.'43'Transla7on'in'Eukaryotes'Ini7a7on'codon'44'Eukaryo7c'transla7on'Ini7a7on'(eIF)'45'40S'Forma7on'of'the'80S'complex'46'60S'40S'Protein'interac7ons'circularize'eukaryo7c'mRNA'47'Synthesis'of'secretory'and'membrane'proteins'Rough're7culum'48'49'50'51'Regula7on'of'protein'synthesis'of)ferriDn)Iron*response'element'binding'protein'X)52'Regula7on'of'protein'synthesis'of)ferriDn)Fe'Ferri7n'Fe'53'Regula7on'of'protein'synthesis'Transferrin.receptor)(protein))Transferrin*receptor'mRNA'54'Regula7on'of'protein'synthesis'Transferrin.receptor)Transferrin*receptor'mRNA'Fe'Fe'X)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
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