Bài giảng Molecular Biology - Chapter 17 The Mechanism of Translation I: Initiation

Molecular BiologyFifth EditionChapter 17The Mechanism of Translation I: InitiationLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.1TranslationTranslation is the process by which ribosomes read the genetic message in mRNA and produce a protein product according to the messageRibosomes are protein factoriesTransfer RNAs (tRNAs) play an important role as adaptors that can bind and amino acid at one end and interact with the mRNA at the other end217.1 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 possible3tRNA 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 tRNA4Two-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 5Aminoacyl-tRNA Synthetase Activity6Dissociation of RibosomesE. coli ribosomes dissociate into subunits at the end of each round of translationRRF and EF-G actively promotes this dissociationIF3 binds to free 30S subunit and prevents reassociation with 50S subunit to form a whole ribosome7Ribosomal Subunit Exchange8Formation of the 30S Initiation ComplexOnce the ribosomomal subunits have been dissociated, the 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 alone9First 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 maturation10N-Formyl-Methionine11Binding 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 RNA12Initiation 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 time13Binding 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 concentrations but GTP is not hydrolyzed in the process1430S Initiation ComplexThe complete 30S initiation complex contains one each:30S ribosomal subunitmRNAfMet-tRNAGTPFactors IF1, IF2, IF315Formation 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 begin16Bacterial Translation InitiationIF1 influences dissociation of 70S ribosome to 50S and 30SBinding IF3 to 30S, prevents subunit reassociationIF1, IF2, and 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 1717.2 Initiation in Eukaryotes Eukaryotic 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 startBasic comparison of initiation between eukaryotes and bacteria18Scanning 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% of the time, most ribosomal subunits bypass 1st AUG scanning for a more favorable one19Translation With a Short ORFSometimes ribosomes can use a short upstream open reading frame: Initiate at an upstream AUGTranslate a short Open Reading Frame (ORF)Continue scanningReinitiate at a downstream AUG20Scanning Model for Translation Initiation21Effects 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 translation22Eukaryotic 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 cap23Translation 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 subunit24Function 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 eIF4F25Function of eIF4A and eIF4BeIF4A 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 mRNA26Function of eIF4GeIF4G is a scaffold 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 translation27Viral Corruption of TranslationInitiation of cellular translation can be corrupted by the picornavirus, poliovirusA viral protease cleaves off the N-terminal domain of eIF4G so that it can no longer recognize caps and capped cellular mRNA is no longer translatedThis cleavage leaves a C-terminal domain called p100 The loss of cap-dependent host protein synthesis in poliovirus infected cells is due to competition by viral RNA for the limitng amount of p10028Functions 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 complexesThey are antagonistic; eIF1 promotes scanning while eIF1A causes the scanning 40S subunit to pause and commit to initiating at the right codon29Principle 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 secondary 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 complex known as mTORC1Target protein for mTOR kinase is a protein called 4E-BP1Once phosphorylated by mTORThis protein dissociates from eIF4EReleases it to participate in active translation initiation 38Phosphorylation of an eIF4E-Binding ProteinAnother target protein for mTOR kinase is S6K1Once phosphorylated by mTORThis protein phosphorylates from eIF4B, which facilitates its association with eIF4A, stimulating initiation of translationIt also phosphorylates PDCD4, which leads to the destruction of PDCD4 and the initiation of translation as PDCD4 is an eIF4A inhibitor 39Repression of Translation by Phosphorylation40Control 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 initiate41Repression 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