5.初级版课件4english vesion英文版-abridgedand intracellular degradation of proteins

1、Chapter39 Biosynthesis & intracellular degradation of proteinsOutlineMajor biomacromolecules involving the translationGeneral Properties of translationDetailed mechanism of translationBacterial translationEukaryotic translationArchaeal translationQuality control of mRNATranslation inhibitorsIntracel

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

3、re translocation. The P (peptidyl-tRNA) site - where peptidyl-tRNA is before peptide bond formation. 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 Prokaryotes) Po

4、lypeptide 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 the intersubuni

5、t 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 grandchildren. The r

6、ibosome 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 monocistronictRNAsSecondary & Tertiary structureIsoacceptor tRNAs- Differe

7、nt 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 tRNAsInitiator tRNA: tRNAf Met & tRNAiMettmRNASingle G3:U70 pair defines specifi

8、cityG:C, A:U or U:G do not work Identity elements in tRNAAla Aminoacyl-tRNA SynthetasesTwo-step Reaction equation: 1) 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

9、 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 charging editing 5) Double sieve ideaIFs, EFs and RFsInitiator factors (IF) Prokaryotes: IF1, IF2 and IF3 Eukaryotes: eIFs El

10、ongation 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: 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 mRNA

11、s 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 hypothesisGeneral Properties of translationThe

12、 Genetic CodeAll the codons have meaning: 61 specify amino acids, and the other 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 rep

13、resenting the same or similar amino acids are similar in sequence: 2nd base 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 t

14、he Genetic CodeExceptions to the genetic codeThe first 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

15、 base G can recognize U or C, and first base I can recognize 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 requir

16、ed to recognize all 61 codons that encode amino acidsAnticodon(base #1)CAGUICodon(base #3)GUC,UA,GU,C,ABacterial TranslationActivation of AAsFormation of fMet-tRNA fMetFormation of other aa-tRNAsInitiationElongationTerminationMetATP/ Mg 2+ +tRNAm MettRNAf MetMet-tRNAmMetMet-tRNAfMetfMet-tRNAfMetN10-

17、FormyltetrahydrofolateTransformylaseAMP/Mg2+ PPiThe same aaRSNo wayAMP/Mg2+ PPiRecognition of the start codon: SD sequence and SD sequenceHow to prove?- Colicin E3 and mutation experimentsFormation of initiation complexBinding of the ribosome 30S subunit with Initiation FactorsBinding of the mRNA an

18、d the fMet-tRNAfMet Binding of the ribosome 50S subunit and release of Initiation FactorsBinding of the ribosome 30S subunit with IFsIF3 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

19、of the 50S subunit prematurely.IF1 assists IF3 in some way.Initiation of translation in BacteriaBinding of the mRNA and the fMet-tRNAfMetIF3 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 positi

20、oned by means of base-pairing between the SD of the 16S rRNA with the SD sequence immediately upstream of the start codon.IF2(GTP) assists the fMet-tRNAfMet to bind to the 30S subunit in the P site.The 30S initiation complex is complete and IF3 can dissociate. Binding of the ribosome 50S subunit and

21、 release of IFsAs IF3 is released, 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 as a timer to ensure that the tRNA is correctly positioned before

22、IF3 dissociates.Once IF2 and IF1 are both released, translation can proceed.Initiation of translation in Bacteria (continued)inactive 70S ribosomeSD sequence30S initiation complex70S initiation complexGDP + PiElongation of translation in Bacteria3 distinct steps to add one amino acid to the growing

23、polypeptide chain. Occurs many times per 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

24、of a new aa-tRNA at the A site -EF-Tu (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 siteFormation of the

25、 new peptide bond (Transpeptidation)Peptide bond formation is simple. It is just a kind of nucleophilic reactionThe peptidyltransferase activity of the ribosome which catalyzes this reaction is located on the 23S rRNA though it will be assisted by some of the ribosomal protein subunits. In other wor

26、ds, 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 translocates along the mRNA thereby moving the new peptidyl-tRNA to the P site and the old (now uncharged)

27、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.GTP is hydrolyzed during translocation and, once again, GTP hydrolysis is required for dissociation o

28、f 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.EF-G and the tRNA-EF-Tu complex are mutually exclusive. The structures of these two are remarkably

29、similar and demonstrate very nicely why these two cannot bind to the ribosome simultaneouslyTranslocation of the RibosomeTerminationA stop codon enters the A site. There are no tRNAs that recognize the stop codons. Rather they are recognized by release factor RF1 (which recognizes the UAA and UAG st

30、op 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 is now the nucleophilic attack agent. The resul

31、t 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 released. IF3 may help this process.Eukaryotic Transla

32、tionDifferences between Bacterial and Eukaryotic TranslationTranslation & transcription are not coupled Ribosomes are larger. The initiating amino acid is still Met, but it is not formylated.Eukaryotic mRNA is capped. This is used as the recognition feature for ribosome binding - not the 18S rRNA.Th

33、e initiation phase of protein synthesis requires over 10 eIFs, one of which is the cap binding protein.The elongation phase requires two eEFsThe termination phase require just a single release factor, eRF. Detailed mechanismActivation of AAsInitiation -Scanning model & Internal entry modelElongation

34、 : Similar to that in Bacteria; eEF-1 is equivalent to EF-Tu and EF-Ts; eEF-2 is equivalent to EF-GTermination- eRF: Similar to that in BacteriaA Comparison of Bacterial and Eukaryotic TranslationThe Scanning modelEukaryotic ribosomes, together with the initiator tRNA (tRNAiMet), generally locate th

35、e 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 AUG and continue to scan for a more favorable one. Sometimes, ribosomes t

36、ranslate a short ORF (open reading frame) by starting at an upstream AUG, then continue scanning and reinitiate at a downstream AUG. Protein Synthesis in ArchaeaAnalyses of most other components of the translation machinery support the close relationship of Archaea to the eukaryotes. In addition to

37、the 11 uniquely eukaryotic ribosomal proteins, eukaryotes have one extra rRNA molecule, the 5.8S rRNA, which is not found in either Bacteria or Archaea.Eukaryotes and Archaea have approximately twice as many translation factors as Bacteria. Those of eukaryotes tend to have more subunits than those o

38、f Archaea, as is the case with many proteins involved in replication. However, the translation factors of Archaea and Eukarya show a high degree of homology. Overall, the components of the translation machinery are more closely related in Archaea and Eukarya than they are to those of Bacteria.The mechanism of protein synthesis in Archaea shares some features with both Bacteria and Eukarya. For example, the mRNAs in both Bacteria and Archaea have sequences complementary to the 3 end of 16S rRNA. that are absent in Eukarya. Conversely, both Archaea and Eukarya insert methionine a