哈佛分子生物学讲义--蛋白质翻译(第三讲)

1、Translation IIIElongation - Big PictureRibosome selects the aa-tRNA in the A-site according to the sequence of the codons (decoding)Ribosome catalyzes peptide-bond formation between the C-terminus of the peptide attached to the tRNA in the P-site and the new aa bound to the tRNA in the A-site - simu

2、ltaneous movement of large particle relative to mRNA and tRNAs by exactly one codonMovement of the tRNAs and mRNA relative to the small particle by one siteErrors in translocation can lead to fameshifting and wrong protein productsRecap: What are A, P and E sites on ribosome Sites in the interface b

3、etween large and small ribosomal subunits where tRNAs reside during protein synthesis A-site is the position where the amino-acid tRNA sits prior to peptide bond formation P-site is the position where is transferred to during peptide-bond formation and where it stays until it moves to the exit site

4、E-site is the spot where the tRNA is placed after it is detached from the growing peptide chain There are A, P and E sites on both subunits movement of mRNA and tRNAs seems to be related to a ratchet-like movement of the two subunits A- and P-sites have been known for long time, the E-site was only

5、discovered in the past decade after ribosome structures have been solved Recently, an F site (factor-binding site) has been discussed as the spot where elongation and termination factors bindSteps of elongationStep 1: new aa-tRNA enters A site - base pairing of anticodon with codon of mRNAincorrect

6、tRNA binds weaker and dissociates before peptide bond can formcorrect tRNA has slower off rate and stays in the average long enough so that peptide bond can form Step 2: Peptide-bond formation and shift of subunitsC-terminus of growing peptide chain detaches from tRNA in P-site and forms peptide bon

7、d with new amino acidconformational changes cause shift of the large subunit placing the two tRNAs into the E- and P-sites of large subunit while they are still in the P- and A- sites of the small particle Step 3: Movement of small subunit relative to mRNAConformational changes place two tRNAs bound

8、 to mRNA into E- and P- sites of small particleRepeat of steps 1 to 3Molecular Biology of theCell, Alberts et al. Role of elongation factors in prokaryotes EF1A (EF-Tu) G protein(GTP/GDP-binding protein) Active when GTP is bound Kd = 10-6 M inactive with GDP is bound Kd = 10-8 M, slow offrate Offrat

9、e is accelerated by EF1B EF1B (EF-Ts) Guanosine nucleotide exchange factor (GEF) for EF1A(EF-Tu) EF2 (EF-G) G protein There is no known GEF for EF-GMore of Elongation FactorsEF1A (EF-Tu) 44 kDa protein, three domains Similar to eukaryotic eEF1A Function is to bind aa-tRNA protect aa-ester bond from

10、hydrolysis Ternary complex of EF1A (EF1A/GTP/aa-tRNA) assists in binding aa-tRNA to the ribosome Large structural change between GTP- and GDP-bound formEF1B (EF-Ts) 30 kDa GEF for EF1AEF2 (EF-G) 77 kDa protein GTP and GDP are bound with similar affinities Therefore, no GEF needed? Higher cellular le

11、vels of GTP than GDP Equilbrium towards the GTP-bound form Catalyzes translocation of the ribosomeElongation cycleMerrick & Nyborg, in “Translational Control in Gene Expression” pp.89 (2000)Elongation cycleTernary complexes (aa-tRNAEF1AGTP) enter ribosome in a “testing phase” where the anticodon

12、 is tested for codon matching, aa-tRNA is in A site, EF1A in a Factor-binding site FMany complexes enter, non-cognate - fast offrates, cognate - slow offratesUpon cognate recognition, EF1AGTP is brought into the GTPase activating center of the ribosome GTP is hydrolyzedEF1AGDP leaves the ribosome. 3

13、CCA end of the aa-tRNA enters the A site of the 50S subunitPeptidyl-transferase center of 50S particle catalyzes peptide-bond formationNow we have the mixed hybrid state:The peptidyl-tRNA is in an A/B state with its anticodon in A site of 30S, whereas the CCA end of acceptor arm is in P site of 50S

14、particleThe newly deacylated tRNA is in a similar P/E hybrid state, with the CCA arm in the E site of 50S, and the anticodon arm in the P site of 30SIn this pre-translocation state, EF2 GTP enters the “ F site” and forces the anticodon arm out of the A site of the small particle and into the P siteD

15、eacylated tRNA is moved fully into the E siteGTP bound to EF2 is hydrolyzed and EF2GDP leaves the ribosomePrecise translocation by one codonEF2 moves the tRNAs, which drag the mRNA with themA, P and E sites are three nucleotides wide and so are the anticodon arms More elongation EF1AGDP is regenerat

16、ed to EF1A GTP by the action of EF1B and loaded with another aa-tRNA Kinetics of these processes have been studied extensively Slowest step is release of EF1A GDP Is there a ratchet-like movement of the two ribosomal particles relative to each other? Attractive model but controversialElongation kine

17、ticsWintermeyer et al. CSH symposia on quantitative biology LXVI, “The Ribosome” p449 (2001)Structural studies of elongation factorsStructures of EF1A GDP and EF1A GDPNP (non-hydrolyzable GTP analog) have been solved There is a large conformational change upon GTP hydrolysis mediated by a helix-to-b

18、-sheet transition in the “ Switch-I region” and a shift of a helix in the “Switch-II region” Both Switch regions contact the other domains and are thus responsible for the large structural changeEF1A EF1B complex has also been solved Mechanism of how the GDP release is facilitated is not yet knownTe

19、rnary complex of EF1A has been solved and exhibits remarkable shape similarity to the structure of EF2EF1A GDP vs EF1A GDPNPEF1AGDPEF1AGDPNPSwitch I Switch I region undergoes a helix-to-sheet transition upon GTP hydrolysis causing a major structural change in the protein. This is thought to be respo

20、nsible for dissociation of the GDP form G/AxxxxGKS/T is the so-called P-loop for nucleotide binding Mg 2+no Mg 2+EF1A EF1B complexEF1A EF1B complex has also been solved (dimer of dimers) Mechanism of how the GDP release is facilitated is not yet known Loss of Mg2+ may be related with GDP dissociatio

21、nG domainDomainsII and IIIEF1AEF1BEF2 mimics shape of ternary complex EF1A aa-tRNAMacromolecular mimicryThe two functional forms are bound to rather similar regionsMakes plausible why EF2 canbind at a similar site as ternary complex and force tRNA over into the next siteEF1A ternary complexEF2G-prot

22、ein domainFrameshifting - Random and Programmed Frameshifting is when the ribosome pauses synthesis and moves back one or two nucleotides (or forward one nucleotide) and continues translationSpontaneous random frameshifting leads to incorrect protein productsProgrammed frameshifting is used by some

23、mRNAs to change the transcript at a particular point within the transcriptProgrammed frameshifting is used to make different products from a single geneExample is E.coli polymerase III; two of the subunits, g and t are made from the same gene Frameshift is thought to be caused byA hairpin loop locat

24、ed after the frameshift positionA mRNA sequence similar to the ribosome-binding site upstream of the frameshift position - stalls ribosome by forming base pairsThe AAG codon for Lys has a weak codon-anticodon interaction of the third base and favors frame shiftingSlippage and bypassing has been obse

25、rved, where longer pieces of mRNA are skippedEukaryotic Elongation Is thought to be very similar to prokaryotic elongation Factors are a larger but mostly homologous eEF1A (50 kDa) eEF1B(abg eEF2 (95 kDa) G protein eEF3 (only found in fungal protein synthesis) Factors have post-translational modific

26、ations Enhanced controlControl of elongationWhy control of elongation?alter rate of synthesis to react upon external stimuliSlow down to conserve energy for other processes (muscle contraction.)Elongation control seems to be faster than initiation control since it doesnt need dissociation of polysom

27、es etc. (both, elongation and initiation control is faster then transcriptional regulation)Slower elongation increases fidelity of translationExamples of elongation controlInsulin has been shown to increase rate of elongationStress caused reduced elongation rates in reticulocytesGlucagon reduces elo

28、ngation rates and cause accumulation ofpolysomesMechanismsPhosphorylation of eEF2 by eEF2 kinase leads to loss of ribosome binding activityAgents that increase cytoplasmic Ca2+ levels cause increased eEF2 phosphorylation5TOP (5 terminal oligopyrimidin tract) is found in a number of mRNAs (eEF1s, Pab

29、p etc.)Termination Termination codon in A site cannot be matched by a ternary complex Release factor RF1 can bind to A site Hydrolysis of peptide bond results in deacylated tRNA in P site RF1 is removed from ribosome in a GTP-dependent reaction involving RF3 Dissociation of 70S/mRNA/deacylated tRNA

30、complex - involvement of IF3Release factorsBacteria have three release factors: RF1 recognizes termination codons UAA and UAG RF2 recognizes termination codons UAA and UGA RF3 stimulates release of RF1 and RF2 from the ribosome with GTP hydrolysisEukaryotes have just two release factors eRF1 has fun

31、ction of RF1 and RF2, recognizes termination codons eRF3 has probabl;y the same role as RF3Ribosome Recycling Factor RRF RRF and eRRF are involved in dissociation of the ribosome, together with IF3 (eIF3) RRF again has a similar overall structure as tRNAeRF1 resembles structure of tRNAWelch et al.,

32、in “Translational control of gene expresssin,pp 467 CSHP 2000The GGQ motif located at the tip of domain 2 is assumed to be a structural counterpart of the tRNA aminoacyl group on the CCA-3 acceptor stemDomain 1 may be equivalent to the anticodon of tRNAStop-codon / release factors factor recognition

33、First and third amino acid of hairpin loop between the helices at the tip of domain 1 are thought to recognize the stop codonsIn bacteria there are two types release factors UAA and UAGUAA and UGATranslational control and cancerCell growth is regulated by a carefully balanced expression of genesCanc

34、erous cells arise when a number of these genes are mutated disturbing the balanced networkStepwise accumulation of mutations that cause malignancy is thought to occur primarily in genes for growth factors, growth-factor receptors Protein kinases involved in signaling pathways Transcription factorsTh

35、e mutations cause dysregulation of cell growth and/or proliferationTransformed cells show a higher rate of protein synthesis Does this contribute to the cause of cancer or is this a consequence of the higher proliferation rate of malignant cells?Hershey and Miyamoto, in “Translational control of gen

36、e expression” CSHP pp637 (2000)Translation initiation factors and cancer elevated levels of eIF4E, eIF4G and eIF4A are observed in several forms of cancer breast cancer (eIF4E), melanoma (eIF4A), lung cancer (eIF4G) elevated eIF2 levels observed in cell transformed with c-myc, v-src and v-abl gene a

37、mplification reported for translation initiation factors in various cancersbeast cancer (eIF4E), lung cancer (eIF4G) transcriptional activation of eIF2a and eIF4E by c-myc the concept of weak and strong mRNAs:- weak mRNAs are poorly translated in resting cells long and structured (GC-rich) 5UTRs (ne

38、ed unwinding) oncogenes and growth factors have often long 5UTRs (ODC, cyclin D1, c-myc) (Kozak, JCB 115, 887-903 (1991)- strong mRNAs are efficiently translated and are found in genes for housekeeping and generally needed proteins downregulation of translation initiation factors may inhibit tumor g

39、rowth translational repression of some oncogenes can be relieved by overexpression of eIF4EMechanisms of translational regulationlong and structured 5UTRadditional initiation codons in 5UTRupstream ORFsIRESs5-TOP (terminal oligopyrimidine) tractstargeted mRNA degradationGrowth factors, cytokinesFGF-

40、2 (IRES)IGF II (long 5-UTR)PDGF (IRES)TGF-b(long 5-UTR)VEGF (IRES)Il-15 (additional AUGs)Hormone receptorsAndrogene receptor (long 5-UTR)Retinoic acid receptor-b2 (uORF)Protein kinasesLck (uORF)c-mos (uORF)pim-1 (uORF)Transcription factorsc-fos (mRNA degradation)c-myc (additional AUGs)BTEB (additional AUGs)C/EBPbTranslationally regulated mRNAs whose gene products are important for cell growthTranslation machineryeIF4EeIF4G (IRES)Enzymes of polyamine biosynthesisODC (long 5-UTR)Ado-Met-DC (uORF)Ornithine aminotransferase (long 5-UTR)Cell cycle and apopt