生物化学原理课件（英文）：Chapter39 Biosynthesis and intracellular degradation of proteins

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1、Chapter39 Biosynthesis and intracellular degradation of proteinsOutlineMajor biomacromolecules involving the translationGeneral Properties of translationDetailed mechanism of translation1.Bacterial translation2.Eukaryotic translation3.Archaeal translationQuality control of mRNATranslation inhibitors

2、Intracellular proteolysisRibosomes as seen with electron microscopeBacterial ribosome model based on Xray diffraction studiesFunctional sites on RibosomeThree sites for tRNA 1.The A (acceptor) site - where aa-tRNAs come in (except the first one) and where peptidyl-tRNA is after peptide bond formatio

3、n 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 translocation. Peptidyl transferase - the active site that catalyzes formation of the peptide bond (23S rRNA in P

4、rokaryotes) Polypeptide exit channel mRNA binding siteThree sites for tRNA on the ribosomeFormation of Polysome The ribosome is a ribozyme?!“ . most 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 t

5、he 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 the modern ribosome is composed of a somewhat geriatric, but functionally vital, RNA scaffold that is propped up and doted upon by its protein grandc

6、hildren. The ribosome Is one colossal enzyme.” James R. Williamson Scripps, Skaggs Inst. Nature 407: 306 Science 289:878 (T.R. Cech)Science289.878.pdf Translation templates-mRNAsBacterial mRNAs Usually polycistronicEukaryotic mRNAs Usually monocistronicSD Sequences recognized by E.coli ribosomestRNA

7、sSecondary & Tertiary structureIsoacceptor tRNAs- Different tRNAs carrying the same aatRNA identity- “2nd genetic code”- Special sequence elements on tRNAs that can be recognized by aaRS and then determine which aa is charged; Positive elements & Negative elementsSome special tRNAs1.Initiator tRNA:

8、tRNAf Met & tRNAiMet2.tmRNA Single G3:U70 pair defines specificity 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 present Six codons for Ser, which are quite different from one another. Six “isoacceptor” tRNAs

9、 It makes sense that the anti-codon loop is not used to recognize tRNASerIdentity elements in tRNASerThree major Differences with other tRNAsStructure of N-formyl-methionyl-tRNAMetAminoacyl-tRNA SynthetasesTwo-step Reaction equation: 1) ATP + amino acid (AA) - AMP-AA + PPi 2) tRNA + AMP-AA - tRNA-AA

10、 + 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 modified and it still works 4) Pre vs post chargi

11、ng editing 5) Double sieve ideaClass I aaRSs Class II aaRSs Class I: Parallel sheet, Rossmann fold nucleotide binding domain conserved motifs: HIGH and KMSKS;all known ones put aminoacyl on 2OH Class II: Anti-parallel sheet, infrequent fold nucleotide binding domain different motifs, less well conse

12、rved: motifs 1, 2 and 3 almost all known ones put aminoacyl on 3OH ( Phe RS 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 doma

13、in 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 doubl

14、e sieve 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

15、Ile minus a terminal 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 si

16、te, so it does not get rejected - the second sieve Valine and smaller aas - e.g. Alanine - are hydrolyzed IFs, EFs and RFsInitiator factors (IF) Prokaryotes: IF1, IF2 and IF3 Eukaryotes: eIFs Elongation factors (EF) Prokaryotes: EF-Tu, EF-Ts and EF-G Eukaryotes: eEF-1 and eEF-2Release Factors (RF) P

17、rokaryotes: RF-1,RF-2 and RF-3 Eukaryotes: eRFWith mRNA as template, tRNA as carrier for aa, ribosome as assembly siteTranslational polarity 1) Extends in N-end C-end how to prove? 2) Reads mRNAs in 5 3Triplet codon 1) How many bases determine one aa? “three determine one” 2) Cracking of the genetic

18、 code 3) Features of the genetic codeRibosomes recognize aa-tRNA just by virtue of the base-pairing interaction between codons and anticodonsWobble hypothesisGeneral Properties of translationRabbit reticulocytes3H-leucineFinish labeling Period at various intervalsPurify completed peptidesLower tempe

19、rature to decrease the translating rateExperiment showing that translation runs from N-end to C-endDigest 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 MatthaeiWorked wi

20、th an in vitro translation system from E. coli Cell-free extractRibosomestRNAsAmino acidsEnzymesATP, GTP+ mRNA = proteinEThey generated RNAs that are homopolymers Eusing the enzyme polynucleotide phosphorylase (catalyzes random synthesis of RNA chains)Deciphering the first word i.e. nNDPs polynucleo

21、tide phosphorylase (NMP)n + nPiEUsing this system they made a polyU mRNA by programming their reaction with UDPEWhen 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

22、 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 pelle

23、t?Deciphering the first word (continued) 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 They repeated the expe

24、riment with other synthetic homopolymer RNAsJPoly-A gave polylysine (AAA = Lys)JPoly-C gave polyproline (CCC = Pro)JPoly-G gave polyglycine (GGG = Gly)Biochemists Break the CodeJWork with nucleotide copolymers (poly (A,C), etc.), revealed some of the codes JGobind Khorana (organic chemist)J-synthesi

25、zed 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 acidsGetting at the Rest

26、 of the CodeRibosome binding assayDevised by Nirenberg and LederTook 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

27、the binding of the tRNA 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 broug

28、ht to the ribosome by that triplet codon UUUAAAPheRibosomeExperiment: 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 for by the triplet codon!Triplet RNANirenberg

29、and LederETernary complexEVery largeECan be captured on a filter19AAs + 14C-Pro and soluble aaRSsRibosome-binding experimentFilter through NC filter which binds ribosome complexesRadioactivity in filtrate19AAs + 14C-Phe and soluble aaRSsFilter through NC filter which binds ribosome complexesRadioact

30、ivity on filterCFinally Marshall Nirenberg and Philip Leder cracked the entire code in 1964 CThey 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 cor

31、respondences in the code CFound that all the codons (except the 3 stop codons) specified an amino acidCThere are 64 codons and 20 amino acids CTherefore amino acids can be encoded by 1 codonGetting at the Rest of the CodeThe Genetic CodeAll the codons have meaning: 61 specify amino acids, and the ot

32、her 3 are nonsense or stop codons The code is unambiguous - 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

33、pyrimidine: usually nonpolar amino acid ; 2nd base purine: usually polar or charged aa The code is not overlapping The base sequence is read from a fixed starting point, with no punctuation Universal & UnusualThe Nature of the Genetic CodeExceptions to the genetic codeExperiment (1962)tRNA-ACAAntico

34、don (recognizes UGU codon, encodes Cys)Cell-free extract amino acids & enzymesCys-tRNA-ACAProtein has CysRNA templateUGUGUGUGUG.Treat w metal catalyst removes thiol groupsAla-tRNA-ACAProtein has AlaRNA templateUGUGUGUGUG.tRNA is charged with CysCharged amino acid is changed chemicallyOnce an aa-tRNA

35、 has been synthesized the amino acid part makes no contribution to accurate translation of the mRNA.More than one codon exist for most amino acids (except Met and Trp)Organism may have a preferred codon for a particular amino acid Codon usage correlates with abundance of tRNAs (preferred codons are

36、represented by abundant tRNAs) Rare tRNAs correspond to rarely used codonsmRNAs containing rare codons experience slow translationCodon BiasThird-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

37、there 61 different anticodons, 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 firs

38、t two bases 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 re

39、cognize U, C or A (I comes from deamination of A) Advantage of wobble: dissociation of tRNA from mRNA is faster and protein synthesis too The Wobble HypothesisCodon- anticodon interactions With these rules a minimum of 31 different tRNAs is required to recognize all 61 codons that encode amino acids

40、Anticodon(base #1)CAGUICodon(base #3)GUC,UA,GU,C,ABacterial TranslationActivation of AAs1.Formation of fMet-tRNA fMet2.Formation of other aa-tRNAsInitiationElongationTerminationMetATP/ Mg 2+ +tRNAm MettRNAf MetMet-tRNAmMetMet-tRNAfMetfMet-tRNAfMetN10-FormyltetrahydrofolateTransformylaseAMP/Mg2+ PPiT

41、he same aaRSAMP/Mg2+ PPiRecognition of the start codon: SD sequence and SD sequenceHow to prove?- Colicin E3 and mutation experimentsFormation of initiation complex1.Binding of the ribosome 30S subunit with Initiation Factors2.Binding of the mRNA and the fMet-tRNAfMet 3.Binding of the ribosome 50S s

42、ubunit and release of Initiation FactorsBinding of the ribosome 30S subunit with IFs1.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 prematurely.2.IF1 assists IF

43、3 in some way.Initiation of translation in Bacteria Binding of the mRNA and the fMet-tRNAfMet1. 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

44、 SD of the 16S rRNA 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. Binding of the ribosome 50S subunit and release of IFs1. As IF3 is release

45、d, the 50S subunit of 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 bot

46、h released, translation can proceed.Initiation of translation in Bacteria (continued)inactive 70S ribosomeSD sequence30S initiation complex70S initiation complexGDP + PiElongation of translation in Bacteria 3 distinct steps to add one amino acid to the growing polypeptide chain. Occurs many times pe

47、r polypeptide, the number of which depends upon the number of mRNA codons or 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 Binding of a new aa-tRNA at the A site -EF-Tu (

48、GTP), EF-Ts Formation of the new peptide bond (Transpeptidation)-23S rRNA Translocation of the Ribosome -EF-G(GTP) Repeat and Repeat until the stop codon enters the A siteElongation of translation in BacteriaBinding of a new aa-tRNA at the A siteAt the start of each cycle, the A site on the ribosome

49、 is empty, the P site contains a peptidyl-tRNA, and the E site contains an uncharged tRNA. EF-Tu (GTP) binds with an aa-tRNA and brings it to the ribosome. Once the correct aa-tRNA is positioned in the ribosome, GTP is hydrolyzed and EF-Tu (GDP) dissociates away from the ribosome.EF-Tu (GDP) is inac

50、tive and cannot function to bind aa tRNAs. In order to recycle EF-Tu, EF-Ts binds to the EF-Tu (GDP) complex to displace the GDP. GTP then, in turn, displaces EF-Ts. There are two ways that EF-Tu functions to ensure that the correct aa-tRNA is in place:1.EF-Tu prevents the aa end of the charged tRNA

51、 from entering the A site on the ribosome. This ensures that codon-anticodon pairing is checked first before the charged tRNA is irreversibly bound in the A site and a new, potentially incorrect, peptide bond is made.2.GTP hydrolysis is SLOW and EF-Tu cannot dissociate from the ribosome until it occ

52、urs. The amount of time prior to GTP hydrolysis allows the final fidelity check to take place. If the anticodon-codon interaction is incorrect, the aa-tRNA simply dissociates and a new one is brought in. Binding of a new aa-tRNA at the A siteKinetic proofreading When a charged tRNA is positioned int

53、o the A-site, the anticodon must base pair with the mRNA codon. If there is an incorrect match, the incorrect aa-tRNA dissociates from the ribosome before GTP hydrolysis occurs. If there is a correct match, GTP hydrolysis occurs and EF-Tu-GDP leaves the ribosome before the cognate aa-tRNA can dissoc

54、iate and EF-Tu-GDP dissociates instead, leaving the correct tRNA on the ribosome. Formation of the new peptide bond (Transpeptidation) Peptide bond formation is simple. It is just a kind of nucleophilic reaction The peptidyltransferase activity of the ribosome which catalyzes this reaction is locate

55、d on the 23S rRNA though it will be assisted by some of the ribosomal protein subunits. In other words, peptidyl transferase is a ribozyme - another example of a catalytic RNA.The ribosomal peptidyl transferase reaction forming a peptide bondTranslocation of the RibosomeFinally, the ribosome translo

56、cates along the mRNA thereby moving the new peptidyl-tRNA to the P site and the old (now uncharged) tRNA, which has just lost its peptidyl chain, to the E site. This step requires the elongation factor, EF-G(GTP). There are 20,000 molecules/cell of EF-G which is the same as the number of ribosomes.G

57、TP is hydrolyzed during translocation and, once again, GTP hydrolysis is required for dissociation of EF-G not for binding.EF-G blocks the binding of aa tRNAs to the A site as well as blocking the binding of RFs. It effectively makes sure that translocation must take place before the cycle continues

58、. The structures of these two are remarkably similar and demonstrate very nicely why these two cannot bind to the ribosome simultaneouslyTranslocation of the RibosomeTermination A stop codon enters the A site. There are no tRNAs that recognize the stop codons. Rather they are recognized by release f

59、actor RF1 (which recognizes the UAA and UAG stop codons) or RF2 (which recognizes the UAA and UGA stop codons). These RFs act at the A site. A third release factor, RF3 (GTP), stimulates the binding of RF1 and RF2. Binding of the release factors alters the peptidyltransferase activity so that water

60、is now the nucleophilic attack agent. The result is hydrolysis of the peptidyl-tRNA and release of the completed polypeptide chain. The uncharged tRNA then dissociates as do the release factors. GTP is hydrolyzed. Finally, the ribosome dissociates into its 30S and 50S subunits and the mRNA is releas

61、ed. IF3 may help this process.Eukaryotic TranslationDifferences between Bacterial and Eukaryotic Translation1.Translation & transcription are not coupled 2.Ribosomes are larger. 3.The initiating amino acid is still Met, but it is not formylated.4.Eukaryotic mRNA is capped. This is used as the recogn

62、ition feature for ribosome binding - not the 18S rRNA.5.The initiation phase of protein synthesis requires over 10 eIFs, one of which is the cap binding protein.6.The elongation phase requires two eEFs7.The termination phase require just a single release factor, eRF. Detailed mechanism1.Activation o

63、f AAs2.Initiation -Scanning model & Internal entry model3.Elongation : Similar to that in Bacteria; eEF-1 is equivalent to EF-Tu and EF-Ts; eEF-2 is equivalent to EF-G4.Termination- eRF: Similar to that in BacteriaA Comparison of Bacterial and Eukaryotic TranslationThe Scanning model Eukaryotic ribo

64、somes, together with the initiator tRNA (tRNAiMet), generally locate the appropriate start codon by binding to the 5 cap of an mRNA and scanning downstream until they find the first AUG in a favorable context. The best context is ACCAUGG. In 5-10% of the cases, the ribosomes will bypass the first AU

65、G and continue to scan for a more favorable one. Sometimes, ribosomes translate a short ORF (open reading frame) by starting at an upstream AUG, then continue scanning and reinitiate at a downstream AUG. It has recently been found that the eukaryotic initiation factor binds not only with other facto

66、rs in the initiation complex but also with PABP (polyA binding protein) which binds to the polyA tail of mRNA.It is though that the binding of to PABP serves as a crticial recruitment step for driving downstream translation.In another sense, however, the binding of to PABP represents a mechanism to ensure that only mature intact mRNAs are translated.Coordinating Protein Synthesis with mRNA SynthesiseIF3 & eIF6eIF1Am7Gm7GATPADP +PieIF-4Initiation of Eukaryotic translationeIF5ATPATP + PiScanningPr