Translation&生物化学讲义课件.pptx
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1、Translation&Post-translational processing,sorting and targetingMajor components 1)Ribosome 2)mRNAs 3)tRNAs 4)aminoacyl-tRNA synthetases(aaRS)5)IFs,EFs and RFsGeneral featuresDetailed MechanismPost-transcriptional ProcessingSorting and TargetingFunctional sites on RibosomeThree sites for tRNA 1)The A
2、(acceptor)site-where aa-tRNAs come in(except the first one)and where peptidyl-tRNA is after peptide bond formation and before translocation.2)The P(peptidyl-tRNA)site-where peptidyl-tRNA is before peptide bond formation.3)The E(exit)site-where the uncharged tRNA from the P site goes after translocat
3、ion.Peptidyl transferase-the active site that catalyzes formation of the peptide bond(23S rRNA in Prokaryotes)Polypeptide exit channel mRNA binding sitesmall subunitlarge subunitmRNAlocationEF-GtRNAStructure of the E.coli RibosomeRibosome cycleRibosome cycleSmall unit Large unitRibosomePolysome+“.mo
4、st biologists did not seriously consider the possibility that RNA could be playing more than a bit part.In the ribosome it has turned out that most of the intersubunit interface is RNA,the peptidyl transferase centre is RNA,and the decoding site and most of the A and P sites are RNA.It appears that
5、the modern ribosome is composed of a somewhat geriatric,but functionally vital,RNA scaffold that is propped up and doted upon by its protein grandchildren.The ribosome Is one colossal enzyme.”James R.Williamson Scripps,Skaggs Inst.Nature 407:306 Science 289:878(T.R.Cech)Science289.878.pdf mRNAsmRNAs
6、Prokaryotic mRNAs Usually polycistronicEukaryotic mRNAs Usually monocistronicShine-Dalgarno Sequences recognized by E.coli ribosomestRNAsSecondary&Tertiary structureIsoacceptor tRNAs Different tRNAs carrying the same aatRNA identity-“2nd genetic code”Special sequence elements on tRNAs that can be re
7、cognized by aaRS and then determine which aa is charged Positive elements&Negative elementsSome special tRNAs 1)Initiator tRNA:tRNAf Met&tRNAiMet 2)Suppressor tRNA 3)tmRNA1)The 5 bases that are identity elements reside in the anticodon(3),G20 in the D loop and A73 near the 3end 2)G20 may be especial
8、ly important in recognition of tRNAPhe since it is not found in any other tRNAIdentity elements in tRNAPhe1)Six codons for Ser,which are quite different from one another.2)Six“isoacceptor”tRNAs3)It makes sense that the anti-codon loop is not used to recognize tRNASerIdentity elements in tRNASer1)Sin
9、gle G3:U70 pair defines specificity2)G:C,A:U or U:G do not work Identity elements in tRNAAla A completely synthetic“microhelix”can be aminoacylated provided that G3:U70 is presentStructure of N-formyl-methionyl-tRNAMetDifferences with other tRNAsAminoacyl-tRNA SynthetasesTwo-step Reaction equation:1
10、)ATP+amino acid(AA)-AMP-AA+PPi 2)tRNA+AMP-AA-tRNA-AA+AMP Classification 1)Class I aaRSs 2)Class II aaRSsProof-reading-quality control at the level of charging 1)The RS is the only place where aa identity is checked 2)The ribosome doesnt care what aa is attached to a tRNA 3)Charged tRNA can be modifi
11、ed and it still works 4)Pre vs post charging editing 5)Double sieve idea Class IParallel sheet,Rossmann fold nucleotide binding domain conserved motifs:HIGH and KMSKS;all known ones put aminoacyl on 2OH Class IIAnti-parallel sheet,infrequent fold nucleotide binding domain different motifs,less well
12、conserved:motifs 1,2 and 3 almost all known ones put aminoacyl on 3OH(PheRS an exception)Proof-reading of aaRSPre-charging editing 1)Sometimes aaRS will activate the wrong aa 2)Binding of cognate tRNA triggers hydrolysis of the aa-AMP instead of charging 3)aa-AMP is transferred to an editing domain
13、that preferentially hydrolyzes incorrect aa-AMP Post-charging editing 1)Sometimes aaRS will activate the wrong aa AND transfer it to the tRNA 2)aa-tRNA is transferred to an editing domain that preferentially hydrolyzes incorrect aa-AMP before the aa-tRNA can be released from the aaRS.The double siev
14、e idea 1)Hard to build the perfect active site 2)activation and editing sites each act as sieves to reject different kinds of errors An Example-IleRSIsoleucine is a large hydrophobic aa.hydrophobic aas larger than Ile cant fit into the activation pocket-the first sieveValine looks like Ile minus a t
15、erminal CH3 group-valine fits the activation pocket Valine leaves a hole in the complex,so Ile still binds better Valine binds well enough to give significant misactivation The editing domain has an active site that fits valine perfectly Ile is too large to fit into the editing site,so it does not g
16、et rejected-the second sieve Valine and smaller aas-e.g.Alanine-are hydrolyzed Experiment(1962)Experiment(1962)tRNA-ACAAnticodon(recognizes UGU codon,encodes Cys)Cell-free extract amino acids&enymesCys-tRNA-ACAProtein has CysRNA templateUGUGUGUGUG.Treat w metal catalyst removes thiol groups(Raney ni
17、ckel)Ala-tRNA-ACAtRNA is charged with CysCharged amino acid is changed chemicallyExperiment(1962)Experiment(1962)tRNA-ACAAnticodon(recognizes UGU codon,encodes Cys)Cell-free extract amino acids&enymesCys-tRNA-ACAProtein has CysRNA templateUGUGUGUGUG.Treat w metal catalyst removes thiol groupsAla-tRN
18、A-ACAProtein has AlaRNA templateUGUGUGUGUG.tRNA is charged with CysCharged amino acid is changed chemicallyOnce an aa-tRNA has been synthesized the amino acid part makes no contribution to accurate translation of the mRNA.IFs,EFs and RFsInitiator factors(IF)Prokaryotes:IF1,IF2 and IF3 Eukaryotes:eIF
19、s Elongation factors(EF)Prokaryotes:EF-Tu,EF-Ts and EF-G Eukaryotes:eEF-1 and eEF-2Release Factors(RF)Prokaryotes:RF-1,RF-2 and RF-3 Eukaryotes:eRFGeneral featuresWith mRNA as template,tRNA as carrier for aa,ribosome as assembly siteTranslational polarity 1)Extends in N-end C-end how to prove?2)Read
20、s mRNAs in 5 3Triplet codon 1)How many bases determine one aa?“three determine one”2)Cracking of the genetic code 3)Features of the genetic codeRibosomes recognize aa-tRNA just by virtue of the base-pairing interaction between codons and anticodonsWobble hypothesisRabbit reticulocytes3H-leucineFinis
21、h labeling Period at various intervalsPurify completed peptidesLower temperature to decrease the translating rateDigest with trpsin,isolate peptides and plot label vs.peptide position Biochemists Break the Code Assignment of codons to their respective amino acidsMarshall Nirenberg and Heinrich Matth
22、aeiWorked with an in vitro translation system from E.coli Cell-free extractRibosomestRNAsAmino acidsEnzymesATP,GTP+mRNA=proteinThey generated RNAs that are homopolymers using the enzyme polynucleotide phosphorylase(catalyzes random synthesis of RNA chains)Deciphering the first word i.e.nNDPs polynuc
23、leotide phosphorylase (NMP)n+nPiUsing this system they made a polyU mRNA by programming their reaction with UDPWhen this was put into the cell-free extract it should be translated into a protein made up of amino acids coded by the codon UUUExperiment:They set up 20 different test tube reactionsEach
24、one was spiked with a different radioactive aaThey programmed each with the poly-U RNAThen recovered the proteins by acid precipitationUnder these conditions the proteins precipitate but the free amino acids do notThen they asked which reaction(out of the 20)has radioactivity in the protein pellet?D
25、eciphering the first word(continued)Biochemists Break the Code Results JMarshall Nirenberg and Heinrich Matthaei showed that poly-U produced polyphenylalanine in a cell-free solution from E.coli.In other words,only the test tube reaction spiked with radioactive Phe generated a radioactive pellet The
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