1. FCH 532 Lecture 19 This material will not be on the exam Chapter 32: Translation

2. Translation The participants Ribosome - ribonucleoprotein enzyme and structural complex 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

3. A comparison of the structures of procaryotic and eucaryotic ribosomes. The ribosome is an allosteric enzyme. The peptidyl-transferase site is the ACTIVE SITE

4. The Prokaryotic Cast (in eukaryotes similar but more complex) Ribosome ~3 x 10 6 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, 2,3Initiation EF-Tu, EF-Ts, EF-G Elongation RF 1, 2, 3, RRFRelease GTP hydrolysis

5. Figure 32-41Ribosomal peptidyl transferase reaction forming a peptide bond. Page 1320

6. Figure 32-43Some translational initiation (Shine- Dalgarno) sequences recognized by E. coli ribosomes. Page 1321 Shine-Dalgarno sequences typically start 10-15 nt upstream of the initiation codon. Are only found in prokaryotes.

7. Figure 32-45Translational initiation pathway in E. coli. Page 1323 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.

8. Page 1327

9. 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

10. 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.

11. cryo-EM

13. Page 1333 EF-Ts EF-Tu-EF-Ts GDP GTP EF-Ts EF-Tu-GTP Aminoacyl-tRNA

14. Page 1335 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). 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

15. Translation Shine-Dalgarno sequence Initiation Elongation Release

16. Page 1322