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
26、y repeated the experiment with other synthetic homopolymer RNAsJPoly-A gave polylysine (AAA=Lys)JPoly-C gave polyproline(CCC=Pro)JPoly-G gave polyglycine(GGG=Gly)Getting at the Rest of the Code JWork with nucleotide copolymers(poly(A,C),etc.),revealed some of the codes JGobind Khorana(organic chemis
27、t)J-synthesized DNA composed of alternating copolymers eg:ACACACACACAC.JThen used RNAP to make RNA from the DNA template eg:UGUGUGUGUGUGUJThis RNA transcript has two possible alternating codons:UGU GUG UGU GUGJIn a translation extract you should get a protein with 2 alternating amino acidsRibosome b
28、inding assay devised by Nirenberg and Leder Took a cell-free translation extract(ribosomes and tRNAs charged with their specific amino acid)Added a synthetic triplet RNA(a codon)eg UUUThey found that addition of that simple triplet RNA to the cell-free extract could stimulate the binding of the tRNA
29、 that recognized that codon to a ribosomeSince the tRNA is covalently linked to the amino acid that is coded for by the codon,therefore that amino acid gets localized to the ribosomeIf they collect the ribosomes from the experiment they can identify which amino acid was brought to the ribosome by th
30、at triplet codon Nirenberg and Leder UUUAAAPheRibosomeTernary complexVery largeCan be captured on a filterExperiment:for each triplet RNA set up 20 reactions,each one spiked with a different radioactive amino acid.Ask which reaction generates radioactivity on the filter.Thats the amino acid coded fo
31、r by the triplet codon!Triplet RNA19AAs+14C-Pro and soluble aaRSsFilter through NC filter which binds ribosome complexesRadioactivity in filtrate19AAs+14C-Phe and soluble aaRSsFilter through NC filter which binds ribosome complexesRadioactivity on filterGetting at the Rest of the Code lFinally Marsh
32、all Nirenberg and Philip Leder cracked the entire code in 1964 lThey showed that trinucleotides bound to ribosomes could direct the binding of specific aminoacyl-tRNAs By using C-14 labeled amino acids with all the possible trinucleotide codes,they elucidated all 64 correspondences in the code lFoun
33、d that all the codons(except the 3 stop codons)specified an amino acidlThere are 64 codons and 20 amino acids lTherefore amino acids can be encoded by 1 codonThe Nature of the Genetic Code All the codons have meaning:61 specify amino acids,and the other 3 are nonsense or stop codons The code is unam
34、biguous-only one amino acid is indicated by each of the 61 codons The code is degenerate-except for Trp and Met,each amino acid is coded by two or more codons Codons representing the same or similar amino acids are similar in sequence 2nd base pyrimidine:usually nonpolar amino acid 2nd base purine:u
35、sually polar or charged aa The code is not overlapping The base sequence is read from a fixed starting point,with no punctuation Universal&UnusualCodon Exceptions:SelenocysteineCodon context can change codon meaning.Several enzymes(for example,glutathione peroxidase,T4-5 deiodinase and formate dehyd
36、rogenase)contain the unusual amino acid selenocysteine They use UGA s in the special context as the codon for Sec.In the absence of selenium,protein synthesis from these mRNAs terminates prematurely.Codon Bias More than one codon exist for most amino acids(except Met and Trp)Organism may have a pref
37、erred codon for a particular amino acid Codon usage correlates with abundance of tRNAs(preferred codons are represented by abundant tRNAs)Rare tRNAs correspond to rarely used codonsmRNAs containing rare codons experience slow translationExamples of codon usage in E.coli and humans GluGAA43.526.4GAG1
38、9.241.6LysAAA36.521.9AAG1235.2ProCCA8.215.4CCC4.320.6CCG23.86.8CCU6.616.1AA Codon E.coliHuman frequency/1000 codonsThird-Base Degeneracy and the Wobble Hypothesis Codon-anticodon pairing is the crucial feature of the reading of the code But what accounts for degeneracy:are there 61 different anticod
39、ons,or can you get by with fewer than 61,due to lack of specificity at the third position?Cricks Wobble Hypothesis argues for the second possibility-the first base of the anticodon(which matches the 3rd base of the codon)is referred to as the wobble position The Wobble Hypothesis The first two bases
40、 of the codon make normal(canonical)H-bond pairs with the 2nd and 3rd bases of the anticodon At the remaining position,less stringent rules apply and non-canonical pairing may occur The rules:first base U can recognize A or G,first base G can recognize U or C,and first base I can recognize U,C or A(
41、I comes from deamination of A)Advantage of wobble:dissociation of tRNA from mRNA is faster and protein synthesis too Codon-anticodon interactions With these rules a minimum of 31 different tRNAs is required to recognize all 61 codons that encode amino acidsAnticodon(base#1)CAGUICodon(base#3)GUC,UA,G
42、U,C,ADetailed mechanism4-stage reaction 1)Activation of AAs 2)Initiation 3)Elongation 4)TerminationIn ProkaryotesIn EukaryotesIn organellesTranslation in Prokaryotes Activation of AAs 1)Formation of fMet-tRNA fMet 2)Formation of Sec-tRNASec 3)Formation of some other aa-tRNAs Initiation Elongation Te
43、rminationMetATP/Mg 2+tRNAm MettRNAf MetMet-tRNAmMetMet-tRNAfMetfMet-tRNAfMetN10-FormyltetrahydrofolateTransformylaseAMP/Mg2+PPiThe same aaRSAMP/Mg2+PPitRNASec +ATP +SerSer RSSer-tRNASecHSe-+ATPSePSec-tRNASecInitiation of translation in Prokaryotes Recognition of the start codon AUG(90%),GUG(8%)and U
44、UG(1%)1)SD sequence and SD sequence How to prove?Colicin E3(大肠杆菌素大肠杆菌素）and mutation experiments 2)Secondary structure of mRNAs Formation of initiation complex 1)Binding of the ribosome 30S subunit with Initiation Factors 2)Binding of the mRNA and the fMet-tRNAfMet 3)Binding of the ribosome 50S subun
45、it and release of Initiation Factors MS2 Phage RNAInitiationlBinding of the ribosome 30S subunit with IFs 1)IF3 promotes the dissociation of the ribosome into its two component subunits.The presence of IF3 permits the assembly of the initiation complex and prevents binding of the 50S subunit prematu
46、rely.2)IF1 assists IF3 in some way.lBinding of the mRNA and the fMet-tRNAfMet 1)IF3 assists the mRNA to bind with the 30S subunit of the ribosome so that the start codon is correctly positioned at the P site of the ribosome.The mRNA is positioned by means of base-pairing between the SD of the 16S rR
47、NA with the SD sequence immediately upstream of the start codon.2)IF2(GTP)assists the fMet-tRNAfMet to bind to the 30S subunit in the P site.3)The 30S initiation complex is complete and IF3 can dissociate.l Binding of the ribosome 50S subunit and release of IFs 1)As IF3 is released,the 50S subunit o
48、f the ribosome binds to complete the initiation complex.Simultaneously,GTP hydrolysis occurs on IF2.Hydrolysis is required for dissociation of IF2.GTP hydrolysis probably serves to ensure that the tRNA is correctly positioned before IF3 dissociates.2)Once IF2 and IF1 are both released,translation ca
49、n proceed.inactive 70S ribosomeSD sequence30S initiation complex70S initiation complexGDP+PiElongation of translation in Prokaryotes3 distinct steps to add one amino acid to the growing polypeptide chain.Occurs many times per polypeptide,the number of which depends upon the number of mRNA codons or
50、amino acids in the protein The Elongation Cycle is similar in prokaryotes and eukaryotes.Fast:15-20 amino acids added per second Accurate:1 mistake every 10,000 amino acids Elongation of translation in ProkaryoteslBinding of a new aa-tRNA at the A site-EF-Tu(GTP),EF-Ts lFormation of the new peptide
