Molecular biology - Chapter 18: The mechanism of translation II: Elongation and termination

Molecular BiologyFourth EditionChapter 18The Mechanism of Translation II: Elongation and TerminationLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.118.1 Direction of Polypeptide Synthesis and mRNA TranslationMessenger RNAs are read in the 5’3’ directionThis is the same direction in which they are synthesizedProteins are made in the aminocarboxyl directionThis means that the amino terminal amino acid is added first2Strategy to Determine Direction of Translation318.2 The Genetic CodeThe term genetic code refers to the set of 3-base code words (codons) in mRNA that represent the 20 amino acids in proteinsBasic questions were answered about translation in the process of “breaking” the genetic code4Nonoverlapping CodonsEach base is part of at most one codon in nonoverlapping codonsIn an overlapping code, one base may be part of two or even three codones5No Gaps in the CodeIf the code contained untranslated gaps or “commas”, mutations adding or subtracting a base from the message might change a few codonsWould still expect ribosome to be back “on track” after the next such commaMutations might frequently be lethalMany cases of mutations should occur just before a comma and have little, if any, effect6Frameshift MutationsFrameshift mutations Translation starts AUGCAGCCAACGInsert an extra base AUXGCAGCCAACGExtra base changes not only the codon in which is appears, but every codon from that point onThe reading frame has shifted one base to the leftCode with commas Each codon is flanked by one or more untranslated bases Commas would serve to set off each codon so that ribosomes recognize it Translation starts AUGZCAGZCCAZACGZInsert an extra base AUXGZCAGZCCAZACGZFirst codon wrong, all others separated by Z, translated normally7Frameshift Mutation Sequences8The Triplet CodeThe genetic code is a set of three-base code words, or codonsIn mRNA, codons instruct the ribosome to incorporate specific amino acids into a polypeptideCode is nonoverlappingEach base is part of only one codonDevoid of gaps or commasEach base in the coding region of an mRNA is part of a codon9Coding Properties of Synthetic mRNAs10Breaking the CodeThe genetic code was brokenUsing: Synthetic messengersSynthetic trinucleotidesThen observing: Polypeptides synthesizedAminoacyl-tRNAs bound to ribosomesThere are 64 codons3 are stop signalsRemainder code for amino acidsThe genetic code is highly degenerate11The Genetic Code12Unusual Base Pairs Between Codon and AnticodonDegeneracy of genetic code is accommodated by:Isoaccepting species of tRNA: bind same amino acid, but recognize different codonsWobble, the 3rd base of a codon is allowed to move slightly from its normal position to form a non-Watson-Crick base pair with the anticodonWobble allows same aminoacyl-tRNA to pair with more than one codon 13Wobble Base PairsCompare standard Watson-Crick base pairing with wobble base pairsWobble pairs are:G-UI-A14Wobble Position15Almost Universal CodeGenetic code is NOT strictly universalCertain eukaryotic nuclei and mitochondria along with at least one bacteriumCodons cause termination in standard genetic code can code for amino acids Trp, GluMitochondrial genomes and nuclei of at least one yeast have sense of codon changed from one amino acid to anotherDeviant codes are still closely related to standard one from which they evolvedGenetic code a frozen accident or the product of evolutionAbility to cope with mutations evolution16Deviations from “Universal” Genetic Code1718.3 The Elongation MechanismElongation takes place in three steps:EF-Tu with GTP binds aminoacyl-tRNA to the ribosomal A sitePeptidyl transferase forms a peptide bond between peptide in P site and newly arrived aminoacyl-tRNA in the A site Lengthens peptide by one amino acid and shifts it to the A siteEF-G with GTP translocates the growing peptidyl-tRNA with its mRNA codon to the P site18Elongation in Translation19A Three-Site Model of the RibosomePuromycin Resembles an aminoacyl-tRNACan bind to the A siteCouple with the peptide in the P siteRelease it as peptidyl puromycinIf peptidyl-tRNA is in the A site, puromycin will not bind to ribosome, peptide will not be releasedTwo sites are defined on the ribosome:Puromycin-reactive site (P)Puromycin unreactive site (A)3rd site (E) for deacylated tRNA bind to E site as exits ribosome20Puromycin Structure and Activity21Protein Factors and Peptide Bond FormationOne factor is T, transferIt transfers aminoacyl-tRNAs to the ribosomeActually 2 different proteinsTu, u stands for unstableTs, s stands for stableSecond factor is G, GTPase activityFactors EF-Tu and EF-Ts are involved in the first elongation stepFactor EF-g participates in the third step22Elongation Step 1Binding aminoacyl-tRNA to A site of ribosome Ternary complex formed from: EF-TuAminoacyl-tRNAGTP Delivers aminoacyl-tRNA to ribosome A site without hydrolysis of GTP Next step: EF-Tu hydrolyzes GTPRibosome-dependent GTPase activityEF-Tu-GDP complex dissociates from ribosome Addition of aminoacyl-tRNA reconstitutes ternary complex for another round of translation elongation23Aminoacyl-tRNA Binding to Ribosome A Site24ProofreadingProtein synthesis accuracy comes from charging tRNAs with correct amino acidsProofreading is correcting translation by rejecting an incorrect aminoacyl-tRNA before it can donate its amino acidProtein-synthesizing machinery achieves accuracy during elongation in two steps25Protein-Synthesizing MachineryTwo steps achieve accuracy:Gets rid of ternary complexes bearing wrong aminoacyl-tRNA before GTP hydrolysisIf this screen fails, still eliminate incorrect aminoacyl-tRNA in the proofreading step before wrong amino acid is incorporated into growing protein chainSteps rely on weakness of incorrect codon-anticodon base pairing to ensure dissociation occurs more rapidly than either GTP hydrolysis or peptide bond formation26Proofreading BalanceBalance between speed and accuracy of translation is delicateIf peptide bond formation goes too fastIncorrect aminoacyl-tRNAs do not have enough time to leave the ribosomeIncorrect amino acids are incorporated into proteinsIf translation goes too slowlyProteins are not made fast enough for the organism to grow successfullyActual error rate, ~0.01% per amino acid is a good balance between speed and accuracy27Elongation Step 2One the initiation factors and EF-Tu have done their jobs, the ribosome has fMet-tRNA in the P site and aminoacyl-tRNA in the A siteNow form the first peptide bondNo new elongation factors participate in this eventRibosome contains the enzymatic activity, peptidyl transferase, that forms peptide bond28Assay for Peptidyl Transferase29Peptide Bond FormationThe peptidyl transferase resides on the 50S ribosomal particleMinimum components necessary for activity are 23S rRNA and proteins L2 and L323S rRNA is at the catalytic center of peptidyl transferase30Elongation Step 3When peptidyl transferase has worked:Ribosome has peptidyl-tRNA in the A siteDeacylated tRNA in the P siteTranslocation, next step, moves mRNA and peptidyl-tRNA one codon’s length through the ribosomePlaces peptidyl-tRNA in the P siteEjects the deacylated tRNAProcess requires elongation factor EF-G which hydrolyzes GTP after translocation is complete31Three-Nucleotide MovementEach translocation event moves the mRNA on codon length, or 3 nt through the ribosome32Role of GTP and EF-GGTP and EF-G are necessary for translocationTranslocation activity appears to be inherent in the ribosomeThis activity can be expressed without EF-G and GTPGTP hydrolysis Precedes translocationSignificantly accelerate translocationNew round of elongation occurs if:EF-G must be released from the ribosomeRelease depends on GTP hydrolysis33GTPases and TranslationSome translation factors harness GTP energy to catalyze molecular motionsThese factors belong to a large class of G proteinsActivated by GTPHave intrinsic GTPase activity activated by an external factor (GAP)Inactivated when they cleave their own GTP to GDPReactivated by another external factor (guanine nucleotide exchange protein) that replaces GDP with GTP34G Protein FeaturesBind GTP and GDPCycle among 3 conformational statesDepends on whether bound to:GDPGTPNeither Conformational state determine activityActivated to carry out functionality when bound to GTPIntrinsic GTPase activity35More G Protein FeaturesGTPase activity stimulated by GTPase activator protein (GAP)When GAP stimulates GTPase cleave GTP to GDPResults in self inactivationReactivation by guanine nucleotide exchange proteinRemoves GDP from inactive G proteinAllows another molecule of GTP to bindExample of guanine nucleotide exchange protein is EF-Ts36Structures of EF-Tu and EF-GThree-dimensional shapes determined by x-ray crystallography: EF-Tu-tRNA-GDPNP ternary complexEF-G-GDP binary complexAs predicted, the shapes are very similar3718.4 TerminationElongation cycle repeats over and overAdds amino acids one at a timeGrows the polypeptide productFinally ribosome encounters a stop codonStop codon signals time for last stepTranslation last step is termination38Termination CodonsThree codons are the natural stop signals at the ends of coding regions in mRNAUAGUAAUGAMutations can create termination codons within an mRNA causing premature termination of translationAmber mutation creates UAGOchre mutation creates UAAOpal mutation creates UGA39Amber Mutation Effects in a Fused Gene40Termination MutationsAmber mutations are caused by mutagens that give rise to missense mutationsOchre and opal mutations do not respond to the same suppressors as do the amber mutationsOchre mutations have their own suppressorsOpal mutations also have unique suppressors41Termination Mutations42Stop Codon SuppressionMost suppressor tRNAs have altered anticodons: Recognize stop codonsPrevent termination by inserting an amino acidAllow ribosome to move on to the next codon43Release FactorsProkaryotic translation termination is mediated by 3 factors:RF1 recognizes UAA and UAGRF2 recognizes UAA and UGARF3 is a GTP-binding protein facilitating binding of RF1 and RF2 to the ribosomeEukaryotes has 2 release factors:eRF1 recognizes all 3 termination codonseRF3 is a ribosome-dependent GTPase helping eRF1 release the finished polypeptide44Release Factor Assays45Dealing with Aberrant TerminationTwo kinds of aberrant mRNAs can lead to aberrant terminationNonsense mutations can occur that cause premature terminationSome mRNAs (non-stop mRNAs) lack termination codonsSynthesis of mRNA was aborted upstream of termination codonRibosomes translate through non-stop mRNAs and then stallBoth events cause problems in the cell yielding incomplete proteins with adverse effects on the cellStalled ribosomes out of actionUnable to participate in further protein synthesis46Non-Stop mRNAsProkaryotes deal with non-stop mRNAs by tmRNA-mediated ribosome rescueAlanyl-tmRNA resembles alanyl-tRNABinds to vacant A site of a ribosome stalled on a non-stop mRNADonates its alanine to the stalled polypeptideRibosome shifts to translating an ORF on the tmRNA (transfer-messenger RNA)Adds another 9 amino acids to the polypeptide before terminatingExtra amino acids target the polypeptide for destructionNuclease destroys non-stop mRNA47Non-Stop mRNAsProkaryotes deal with non-stop mRNAs by tmRNA-mediated ribosome rescuetmRNA are about 300 nt long5’- and 3’-ends come together to form a tRNA-like domain (TLD) resembling a tRNA48Eukaryotic Aberrant TerminationEukaryotes do not have tmRNAEukaryotic ribosomes stalled at the end of the poly(A) tail contain 0 – 3 nt of poly(A) tailThis stalled ribosome state is recognized by carboxyl-terminal domain of a protein called Ski7pSki7p also associates tightly with cytoplasmic exosome, cousin of nuclear exosomeNon-stop mRNA recruit Ski7p-exosome complex to the vacant A siteSki complex is recruited to the A siteExosome, positioned just at the end of non-stop mRNA, degrades that RNAAberrant polypeptide is presumably destroyed49Exosome-Mediated DegradationThis stalled ribosome state is recognized by carboxyl-terminal domain of a protein called Ski7pSki7p also associates tightly with cytoplasmic exosome, cousin of nuclear exosomeNon-stop mRNA recruit Ski7p-exosome complex to the vacant A siteSki complex is recruited to the A site50Premature TerminationEukaryotes deal with premature termination codons by 2 mechanisms:NMD (nonsense-mediated mRNA decay)Mammalian cells use a downstream destabilizing elementYeast cells appear to recognize a premature stop codon NAS (nonsense-associated altered splicing)Senses a stop codon in the middle of a reading frameChanges the splicing pattern so premature stop codon is spliced out of mature mRNABoth mechanisms require Upf151Mammalian NMDNMD in mammalian cells involves a downstream destabilizing elementUpf1Upf2Bind to mRNA at exon-exon junction that measures distance to a stop codonCodon far enough upstreamLooks like a stop codonActivates downstream destabilizing element to degrade mRNA 52Yeast NMDYeast cells appear to recognize a premature stop codon by the absence of a normal 3’-UTR or poly (A) nearbyRibosome stopping at premature stop codon moves to an upstream AUGThis may mark the mRNA for destruction53NAS and NMD Models54Use of Stop Codons to Insert Unusual Amino AcidsUnusual amino acids are incorporated into growing polypeptides in response to termination codonsSelenocysteine uses a special tRNA Anticodon for UGA codonCharged with serine then converted to selenocysteineSelenocysteyl-tRNA escorted to ribosome by special EF-TuPyrrolysine uses a special tRNA synthetase that joins preformed pyrrolysine with a special tRNA having an anticodon recognizing UAG5518.5 PosttranslationTranslation events do not end with terminationProteins must fold properlyRibosomes need to be released from mRNA and engage in further translation roundsFolding is actually a cotranslational event occurring as nascent polypeptide is being made56Folding Nascent ProteinsMost newly-made polypeptides do not fold properly alonePolypeptides require folding help from molecular chaperonesE. coli cells use a trigger factorAssociates with the large ribosomal subunitCatches the nascent polypeptide emerging from ribosomal exit tunnel in a hydrophobic basket to protect from waterArchaea and eukaryotes lack trigger factor, use freestanding chaperones57Release of Ribosomes from mRNARibosomes do not release from mRNA spontaneously after terminationHelp is required from ribosome recycling factor (RRF) and EF-GRRF resembles a tRNABinds to ribosome A siteUses a position not normally taken by a tRNACollaborates with EF-G in releasing either 50S ribosome subunit or whole ribosome 58