Transcript Lecture 27
FCH 532 Lecture 19 This material will not be on the exam Chapter 32: Translation Translation Ribosome - ribonucleoprotein enzyme and structural complex The participants mRNA - carries the DNA information encoding the protein tRNAs - decoders of mRNA amino acids and N-formylmethionine Translation initiation Initiation complex formation (30S ribosome subunit-mRNA-fMet-tRNA) Elongation Binding charged tRNA RNA in large ribosomal subunit is the enzyme in peptide bond formation Translocation Termination Stop codons Release factors A comparison of the structures of procaryotic and eucaryotic ribosomes. The ribosome is an allosteric enzyme. The peptidyl-transferase site is the ACTIVE SITE The Prokaryotic Cast (in eukaryotes similar but more complex) Ribosome ~3 x 106 Da, 250 Å (50S + 30S = 70S) The ribosome provides the structure necessary for translation and catalyzes the reaction What else is needed? Factors: • IF 1, 2,3 Initiation • EF-Tu, EF-Ts, EF-G Elongation • RF 1, 2, 3, RRF Release • GTP hydrolysis Page 1320 Figure 32-41 Ribosomal peptidyl transferase reaction forming a peptide bond. Page 1321 Figure 32-43 Some translational initiation (ShineDalgarno) sequences recognized by E. coli ribosomes. Shine-Dalgarno sequences typically start 10-15 nt upstream of the initiation codon. Are only found in prokaryotes. Page 1323 Figure 32-45 Translational initiation pathway in E. coli. • 50S and 30S associated. • IF3 binds to 30S, causes release of 50S. • mRNA, IF2-GTP (ternary complex), fMet-tRNA and IF1 bind 30S. • IF1 and IF2 are released followed by binding of 50S. • IF2 hydrolyzes GTP and poises fMet tRNA in the P site. Page 1327 Defining tRNA Binding Sites in Different functional States GENERATE RIBOSOMES IN THE FOLLOWING STATES: A (Aminoacyl): EF-Tu.GTP dependent; mRNA dependent; occupied P Site P (Peptidyl): Reactive with Puromycin (Pm) E (Exit): Deacylated tRNA MONITOR BY CHEMICAL FOOTPRINTING: 30S A site protections: 50S P site protections (also X-linkers, EDTA-FeII) Looking at footprint pre and post peptide bond, translocation The data didn't fit into a simple 2 site model HYBRID STATES HAD TO BE INVOKED tRNA movement occurs independently on 2 subunits via 6 hybrid states. 1. A/T --> 2. A/A --> 3. A/P --> 4. P/P --> 5. P/E --> 6. E In this model the tRNA would "ratchet" its way through the ribosome undergoing 50° rotations along its longitudinal axis from A to P. This model has received support from EM and X-ray studies. cryo-EM Aminoacyl-tRNA EF-Tu-GTP EF-Ts GTP EF-Tu-EF-Ts Page 1333 EF-Ts GDP RF-1 = UAA RF-2 = UAA and UGA Cannot bind if EF-G is present. RF-3-GTP binds to RF1 after the release of the polypeptide. Hydrolysis of GTP on RF-3 facilitates the release of RF-1 (or RF-2). Page 1335 EF-G-GTP and ribosomal recycling factor (RRF)-bind to A site. Release of GDP-RF-3 EF-G hydrolyzes GTP -RRF moves to the P site to displace the tRNA. RRF and EF-G-GDP are released yielding inactive 70S Translation • • • • Shine-Dalgarno sequence Initiation Elongation Release Page 1322