1、免疫學(xué)信息網(wǎng) ,Translation III,免疫學(xué)信息網(wǎng) ,Elongation - Big Picture,Ribosome 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 i

2、n the A-site - simultaneous movement of large particle relative to mRNA and tRNAs by exactly one codon Movement of the tRNAs and mRNA relative to the small particle by one site Errors in translocation can lead to fameshifting and wrong protein products,免疫學(xué)信息網(wǎng) ,Recap: What are A, P and E sites on rib

3、osome,Sites in the interface between 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 un

4、til it moves to the exit site 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 l

5、ong time, the E-site was only 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 bind,免疫學(xué)信息網(wǎng) ,Steps of elongation,Step 1: new aa-tRNA enters A site - base pairing o

6、f anticodon with codon of mRNA incorrect tRNA binds weaker and dissociates before peptide bond can form correct 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 subunits C-terminus of growing peptide chain detache

7、s from tRNA in P-site and forms peptide bond with new amino acid conformational 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 mRNA

8、 Conformational changes place two tRNAs bound to mRNA into E- and P-sites of small particle Repeat of steps 1 to 3,Molecular Biology of theCell, Alberts et al.,免疫學(xué)信息網(wǎng) ,Role of elongation factors in prokaryotes,EF1A (EF-Tu) G protein(GTP/GDP-binding protein) Active when GTP is bound Kd = 10-6 M inact

9、ive with GDP is bound Kd = 10-8 M, slow offrate Offrate 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-G,免疫學(xué)信息網(wǎng) ,More of Elongation Factors,EF1A (EF-Tu) 44 kDa protein, three domains Similar to eukaryot

10、ic eEF1A Function is to bind aa-tRNA protect aa-ester bond from 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 form EF1B (EF-Ts) 30 kDa GEF for EF1A EF2 (EF-G) 77 kDa protein GTP and GDP are bound wi

11、th similar affinities Therefore, no GEF needed? Higher cellular levels of GTP than GDP Equilbrium towards the GTP-bound form Catalyzes translocation of the ribosome,免疫學(xué)信息網(wǎng) ,Elongation cycle,Merrick two of the subunits, g and t are made from the same gene Frameshift is thought to be caused by A hairp

12、in loop located after the frameshift position A mRNA sequence similar to the ribosome-binding site upstream of the frameshift position - stalls ribosome by forming base pairs The AAG codon for Lys has a weak codon-anticodon interaction of the third base and favors frame shifting Slippage and bypassi

13、ng has been observed, where longer pieces of mRNA are skipped,免疫學(xué)信息網(wǎng) ,Eukaryotic Elongation,Is thought to be very similar to prokaryotic elongation Factors are a larger but mostly homologous eEF1A (50 kDa) eEF1B(a eEF2 (95 kDa) G protein eEF3 (only found in fungal protein synthesis) Factors have pos

14、t-translational modifications Enhanced control,免疫學(xué)信息網(wǎng) ,Control of elongation,Why control of elongation? alter rate of synthesis to react upon external stimuli Slow down to conserve energy for other processes (muscle contraction.) Elongation control seems to be faster than initiation control since it

15、 doesnt need dissociation of polysomes etc. (both, elongation and initiation control is faster then transcriptional regulation) Slower elongation increases fidelity of translation Examples of elongation control Insulin has been shown to increase rate of elongation Stress caused reduced elongation ra

16、tes in reticulocytes Glucagon reduces elongation rates and cause accumulation ofpolysomes Mechanisms Phosphorylation of eEF2 by eEF2 kinase leads to loss of ribosome binding activity Agents that increase cytoplasmic Ca2+ levels cause increased eEF2 phosphorylation 5TOP (5 terminal oligopyrimidin tra

17、ct) is found in a number of mRNAs (eEF1s, Pabp etc.),免疫學(xué)信息網(wǎng) ,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 i

18、nvolving RF3 Dissociation of 70S/mRNA/deacylated tRNA complex - involvement of IF3,免疫學(xué)信息網(wǎng) ,Release factors,Bacteria 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

19、 hydrolysis Eukaryotes have just two release factors eRF1 has function of RF1 and RF2, recognizes termination codons eRF3 has probabl;y the same role as RF3 Ribosome Recycling Factor RRF RRF and eRRF are involved in dissociation of the ribosome, together with IF3 (eIF3) RRF again has a similar overa

20、ll structure as tRNA,免疫學(xué)信息網(wǎng) ,eRF1 resembles structure of tRNA,Welch et al., in “Translational control of gene expresssin,pp 467 CSHP 2000,The 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 stem Domain 1 may be equi

21、valent to the anticodon of tRNA,免疫學(xué)信息網(wǎng) ,Stop-codon / release factors factor recognition,First and third amino acid of hairpin loop between the helices at the tip of domain 1 are thought to recognize the stop codons In bacteria there are two types release factors,UAA and UAG,UAA and UGA,免疫學(xué)信息網(wǎng) ,Trans

22、lational control and cancer,Cell growth is regulated by a carefully balanced expression of genes Cancerous cells arise when a number of these genes are mutated disturbing the balanced network Stepwise accumulation of mutations that cause malignancy is thought to occur primarily in genes for growth f

23、actors, growth-factor receptors Protein kinases involved in signaling pathways Transcription factors The mutations cause dysregulation of cell growth and/or proliferation Transformed cells show a higher rate of protein synthesis Does this contribute to the cause of cancer or is this a consequence of

24、 the higher proliferation rate of malignant cells?,Hershey and Miyamoto, in “Translational control of gene expression” CSHP pp637 (2000),免疫學(xué)信息網(wǎng) ,Translation initiation factors and cancer,elevated levels of eIF4E, eIF4G and eIF4A are observed in several forms of cancer breast cancer (eIF4E), melanoma

25、 (eIF4A), lung cancer (eIF4G) elevated eIF2 levels observed in cell transformed with c-myc, v-src and v-abl gene amplification reported for translation initiation factors in various cancers beast cancer (eIF4E), lung cancer (eIF4G) transcriptional activation of eIF2a and eIF4E by c-myc the concept o

26、f weak and strong mRNAs: - weak mRNAs are poorly translated in resting cells long and structured (GC-rich) 5UTRs (need 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 gene

27、s for housekeeping and generally needed proteins downregulation of translation initiation factors may inhibit tumor growth translational repression of some oncogenes can be relieved by overexpression of eIF4E,免疫學(xué)信息網(wǎng) ,Mechanisms of translational regulation,long and structured 5UTR additional initiati

28、on codons in 5UTR upstream ORFs IRESs 5-TOP (terminal oligopyrimidine) tracts targeted mRNA degradation,免疫學(xué)信息網(wǎng) ,Growth factors, cytokines FGF-2 (IRES) IGF II (long 5-UTR) PDGF (IRES) TGF-b (long 5-UTR) VEGF (IRES) Il-15 (additional AUGs) Hormone receptors Androgene receptor (long 5-UTR) Retinoic aci

29、d receptor-b2 (uORF) Protein kinases Lck (uORF) c-mos (uORF) pim-1 (uORF) Transcription factors c-fos (mRNA degradation) c-myc (additional AUGs) BTEB (additional AUGs) C/EBPb,Translationally regulated mRNAs whose gene products are important for cell growth,Translation machinery eIF4E eIF4G (IRES) En

30、zymes of polyamine biosynthesis ODC (long 5-UTR) Ado-Met-DC (uORF) Ornithine aminotransferase (long 5-UTR) Cell cycle and apoptosis CDK4 (long 5-UTR) Cyclin D1 (long 5-UTR) p27Kip1 MDM-2 p53 (long 5-UTR) Bcl-2 (uORF) Bax (uORF) Fas/Apo-1 (uORF),免疫學(xué)信息網(wǎng) ,Mechanisms of translational control -uORF,Upstream open reading frames (uORF) make translation very sensitive to the cellular concentration of eIF2 ternary complex (studied in detail for GCN4) Due to details of scanning - scanning is still poorly understood When and why