Molecular biology - Chapter 17: the mechanism of translation I: Initiation

Molecular BiologyFourth EditionChapter 17The Mechanism of Translation I: InitiationLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.117.1 Initiation of Translation in BacteriaTwo important events must occur before translation initiation can take placeGenerate a supply of aminoacyl-tRNAsAmino acids must be covalently bound to tRNAsProcess of bonding tRNA to amino acid is called tRNA chargingDissociation of ribosomes into their two subunitsThe cell assembles the initiation complex on the small ribosomal subunitThe two subunits must separate to make assembly possible2tRNA ChargingAll tRNAs have same 3 bases at 3’-end (CCA)Terminal adenosine is the target for charging with amino acidAmino acid attached by ester bond between Its carboxyl group 2’- or 3’-hydroxyl group of terminal adenosine of tRNA3Two-Step ChargingAminoacyl-tRNA synthetases join amino acids to their cognate tRNAsThis is done in a two-step reaction:Begins with activation of the amino acid with AMP derived from ATPIn the second step, the energy from the aminoacyl-AMP is used to transfer the amino acid to the tRNA 4Aminoacyl-tRNA Synthetase Activity5Dissociation of RibosomesE. coli ribosomes dissociate into subunits at the end of each round of translationIF1 actively promotes this dissociationIF3 binds to free 30S subunit and prevents reassociation with 50S subunit to form a whole ribosome6Ribosomal Subunit Exchange7Formation of the 30S Initiation ComplexWhen ribosomes have been dissociated into 50S and 30S subunits, cell builds a complex on the 30S subunit:mRNAAminoacyl-tRNAInitiation factorsIF3 binds by itself to 30S subunitIF1 and IF2 stabilize this bindingIF2 can bind alone, but is stabilized with help of IF1 and IF3IF1 does not bind alone8First Codon and the First Aminoacyl-tRNAProkaryotic initiation codon is:Usually AUGCan be GUGRarely UUGInitiating aminoacyl-tRNA is N-formyl-methionyl-tRNAN-formyl-methionine (fMet) is the first amino acid incorporated into a polypeptideThis amino acid is frequently removed from the protein during maturation9N-Formyl-Methionine10Binding mRNA to the 30S Ribosomal SubunitThe 30S initiation complex is formed from a free 30S ribosomal subunit plus mRNA and fMet-tRNABinding between the 30S prokaryotic ribosomal subunit and the initiation site of a message depends on base pairing betweenShort RNA sequenceShine-Dalgarno sequenceUpstream of initiation codon Complementary sequence3’-end of 16S RNA11Initiation Factors and 30S SubunitBinding of the Shine-Dalgarno sequence with the complementary sequence of the 16S rRNA is mediated by IF3Assisted by IF1 and IF2All 3 initiation factors have bound to the 30S subunit at this time12Binding of fMet-tRNA to the 30S Initiation ComplexIF2 is the major factor promoting binding of fMet-tRNA to the 30S initiation complexTwo other initiation factors also play an important supporting roleGTP is also required for IF2 binding at physiological IF2 concentrationsThe GTP is not hydrolyzed in the process1330S Initiation ComplexThe complete 30S initiation complex contains one each:30S ribosomal subunitmRNAfMet-tRNAGTPFactors IF1, IF2, IF314Formation of the 70S Initiation ComplexGTP is hydrolyzed after the 50S subunit joins the 30S complex to form the 70S initiation complexThis GTP hydrolysis is carried out by IF2 in conjunction with the 50S ribosomal subunitHydrolysis purpose is to release IF2 and GTP from the complex so polypeptide chain elongation can begin15Bacterial Translation InitiationIF1 influences dissociation of 70S ribosome to 50S and 30SBinding IF3 to 30S, prevents subunit reassociationIF1, IF2, GTP bind alongside IF3Binding mRNA to fMet-tRNA forming 30S initiation complexCan bind in either orderIF2 sponsors fMet-tRNAIF3 sponsors mRNABinding of 50S with loss of IF1 and IF3IF2 dissociation and GTP hydrolysis 1617.2 Initiation in EukaryotesEukaryotic Begins with methionineInitiating tRNA not same as tRNA for interior No Shine-DalgarnomRNA have caps at 5’end BacterialN-formyl-methionineShine-Dalgarno sequence to show ribosomes where to start17Scanning Model of InitiationEukaryotic 40S ribosomal subunits locate start codon by binding to 5’-cap and scanning downstream to find the 1st AUG in a favorable contextKozak’s Rules are a set of requirementsBest context uses A of ACCAUGG as +1:Purine in -3 positionG in +4 position5-10% cases ribosomal subunits bypass 1st AUG scanning for more favorable one18Translation With a Short ORFRibosomes can use a short upstream open reading frame: Initiate at an upstream AUGTranslate a short Open Reading FrameContinue scanningReinitiate at a downstream AUG19Scanning Model for Translation Initiation20Effects of mRNA Secondary StructureSecondary structure near the 5’-end of an mRNA can have either positive or negative effectsHairpin just past an AUG can force a pause by ribosomal subunit and stimulate translationVery stable stem loop between cap and initiation site can block scanning and inhibit translation21Eukaryotic Initiation FactorsBacterial translation initiation requires initiation factors as does eukaryotic initiation of translationEukaryotic system is more complex than bacterialScanning processFactors to recognize the 5’-end cap22Translation Initiation in EukaryotesEukaryotic initiation factors and general functions:eIF2 binds Met-tRNA to ribosomeseIF2B activates eIF2 replacing its GDP with GTPeIF1 and eIF1A aid in scanning to initiation codoneIF3 binds to 40S ribosomal subunit, inhibits reassociation with 60S subuniteIF4 is a cap-binding protein allowing 40S subunit to bind 5’-end of mRNAeIF5 encourages association between 60S ribosome subunit and 48S complexeIF6 binds to 60S subunit, blocks reassociation with 40S subunit23Function of eIF4eIF4 is a cap-binding proteinThis protein is composed of 3 parts:eIF4E, 24-kD, has actual cap binding activityeIF4A, a 50-kD polypeptideeIF4G is a 220-kD polypeptideThe complex of the three polypeptides together is called eIF4F24Function of eIF4A and eIF4BeIF4A Has an RNA helicase activityThis activity unwinds hairpins found in the 5’-leaders of eukaryotic mRNAUnwinding activity is ATP dependenteIF4B Has an RNA-binding domainCan stimulate the binding of eIF4A to mRNA25Function of eIF4GeIF4G is an adaptor protein capable of binding to other proteins including:eIF4E, cap-binding proteineIF3, 40S ribosomal subunit-binding proteinPab1p, a poly[A]-binding proteinInteracting with these proteins lets eIF4G recruit 40S ribosomal subunits to mRNA and stimulate translation26Structure and Function of eIF3eIF3 is a 5-lobed protein that binds at the same site to: eIF4GProminent part of viral IRESThis explains how the IRES can substitute for 40S ribosomal subunit to mRNACryo-EM studies have produced a model for the eIF3-IRES-40S complex explaining how eIF3 prevents premature 40S-60S association27Prevention of Premature 40S-60S AssociationeIF3 blocks key contact point between subunits 40S and 60SeIF4G, so also eIF4E, locate close to the cap on an mRNA bound to 40S ribosomal particleeIF4 would be in position to cap-bind28Functions of eIF1 and eIF1AeIF1 and eIF1A act synergistically to promote formation of a stable 48S complex involving:Initiation factorsMet-tRNA40S ribosomal subunits bound at initiation codon of mRNAeIF1 and eIF1A act by Dissociating improper complexes between 40S subunits and mRNAEncouraging formation of stable 48S complexes29Principle of the Toeprint AssaySource: Adapted from Jackson, R., J. G. Sliciano, Cinderella factors have a ball, Nature 394:830, 1998.30Functions of eIF5 and eIF5BeIF5B is homologous to prokaryotic factor IF2Binds GTPUses GTP hydrolysis to promote its own dissociation from ribosomePermits protein synthesis to beginStimulates association of 2 ribosomal subunitsDiffers from IF2 as eIF5B cannot stimulate binding of initiating aminoacyl-tRNA to small ribosomal subuniteIF5B works with eIF53117.3 Control of InitiationGiven the amount of control at the transcriptional and posttranscriptional levels, why control gene expression at translational level?Major advantage = speedNew gene products can be produced quickly Simply turn on translation of preexisting mRNAValuable in eukaryotesTranscripts are relatively longTake correspondingly long time to makeMost control of translation happens at the initiation step32Bacterial Translational ControlMost bacterial gene expression is controlled at transcription levelMajority of bacterial mRNA has a very short lifetimeOnly 1 to 3 minutesAllows bacteria to respond quickly to changing circumstancesDifferent cistrons on a polycistronic transcript can be translated better than others33Shifts in mRNA Secondary StructuremRNA secondary structure can govern translation initiationReplicase gene of the MS2 class of phagesInitiation codon is buried in secondary structure until ribosomes translating the coat gene open up the structureHeat shock sigma factor, s32 of E. coliRepressed by secondary structure that is relaxed by heatingHeat can cause an immediate unmasking of initiation codons and burst of synthesis34Proteins/mRNAs Induce mRNA Secondary Structure ShiftsSmall RNAs with proteins can affect mRNA 2° structure to control translation initiationRiboswitches can be used to control translation initiation via mRNA 2° structure5’-untranslated region of E. coli thiM mRNA contain a riboswitchThis includes an aptamer that binds thiamine and its metaboliteThiamine phosphateThiamine pyrophosphate (TTP)35Activation of mRNA TranslationWhen TPP abundantBinds aptamerCauses conformational shift in mRNATies up Shine-Dalgarno in 2° structureShift hides the SD sequence from ribosomesInhibits translation of mRNASaves energy as thiM mRNA encodes an enzyme needed to produce more thiamine and TPP36Eukaryotic Translational ControlEukaryotic mRNA lifetimes are relatively long, so there is more opportunity for translation control than in bacteriaeIF2 a-subunit is a favorite target for translation controlHeme-starved reticulocytes activate HCR (heme-controlled repressor) Phosphorylates eIF2aInhibit initiationVirus-infected cells have another kinase, DAIPhosphorylates eIF2aInhibits translation initiation37Phosphorylation of an eIF4E-Binding ProteinInsulin and a number of growth factors stimulate a pathway involving a protein kinase known as mTORmTOR kinase’s target proteinPHAS-1 (rat)4E-BP1 (human)Once phosphorylated by mTORThis protein dissociates from eIF4EReleases it to participate in active translation initiation 38Repression of Translation by Phosphorylation39Stimulation of Translation by Phosphorylation of PHAS-140Control of Translation Initiation by MaskinIn Xenopus oocytes, Maskin binds to eIF4E and to CPEB (cytoplasmic polyadenylation element binding-protein)Maskin bound to eIF4E, cannot bind to eIF4G, translation is now inhibitedUpon activation of oocytesCPEB is phosphorylatedPolyadenylation is stimulateMaskin dissociates from eIF4EWhen Maskin is no longer attachedeIF4E able to associate with eIF4GTranslation can initiate41Stimulation by an mRNA-Binding ProteinFerritin mRNA translation is subject to induction by ironInduction seems to work as follows:Repressor protein (aconitase apoprotein) binds to stem loop iron response element (IRE)Binding occurs near 5’-end of the 5’-UTR of the ferritin mRNAIron removes this repressor and allows mRNA translation to proceed42Blockage of Translation Initiation by an miRNAThe let-7 miRNA shifts the polysomal profile of target mRNAs in human cells toward smaller polysomesThis miRNA blocks translation initiation in human cellsTranslation initiation that is cap-independent due to presence of an IRES, or a tethered initiation factor, is not affected by let-7 miRNAThis miRNA blocks binding of eIF4E to the cap of target mRNAs in the human cell43