1. Protein Synthesis 1 Major topics covered: The genetic code tRNA: aminoacylation and base-pairing Ribosome structure/function: prokaryotic versus eukaryotic related text: Biochemistry Garret and Grisham, 4 th ed. Chapter 30 contact info: David A. Schneider, Ph.D. Department of Biochemistry and Molecular Genetics dschneid@uab.edu office #: 934-4781

2. The Central Dogma of Biology: DNA RNA protein

3. The Central Dogma of Biology: DNA RNA protein Other macromolecules

4. The Central Dogma of Biology: DNA RNA protein

5. A major molecular problem: How do you take a 4-base DNA/RNA code and interpret the instructions to build proteins from a 20 amino acid pool?

6. A major molecular problem: How do you take a 4-base DNA/RNA code and interpret the instructions to build proteins from a 20 amino acid pool? rephrase: How do you translate the 4-base DNA/RNA language into appropriate proteins?

7. Francis Crick proposed/predicted the Adaptor Hypothesis – …the RNA of the microsomal particles, regularly arranged, is the template – …whatever went into the template in a specific way did so by forming hydrogen bonds – …the amino acid is carried to the template by an adaptor... – such adaptors…might contain nucleotides – …a separate enzyme would be required to join each adaptor to its own amino acid… – …the specificity required to distinguish between … isoleucine and valine would be provided by these enzymes Currently Known As: mRNA Codon-Anticodon Interactions Aminoacyl-tRNA tRNA Aminoacyl-tRNA Synthetase Editing by Aminoacyl-tRNA synthetases Crick, FHC. 1958. Symp. Soc. Exp. Biol. 12: 138-163. Cricks Predictions:

8. A visual model for the adapter hypothesis

9. Thus: Genes are codes (recipes, in a way) RNA polymerases copy the code into useful templates crack correctly Translation (a collaboration of tRNA and ribosomes) must crack the code correctly

10. The genetic code uses 3-base codons to generate 64 possible codon:anticodon interactions (from the 4-base DNA/RNA sequence)

11. Different amino acids are encoded by one or more codons

12. tRNAs are the adapters that crack the triplet code and mediate the codon:anticodon pairing

13. Translation (on the surface) is very simple: Charged tRNAs bind to the appropriate codons Put a bunch in a row, according to the recipe in the mRNA Bind all the amino acids together and, Wa-La!

14. Translation (on the surface) is very simple: Charged tRNAs bind to the appropriate codons Put a bunch in a row, according to the recipe in the mRNA Bind all the amino acids together and, Wa-La! There are at least 3 major issues: 1.Proper amino acid must be attached to every tRNA 2.Proper binding of tRNA (anticodon) to mRNA (codon) must occur 3.Triplet code must be interpreted in the proper frame

15. Problem #1 Charging of the tRNA (ie. aminoacylation)

16. The amino acid is covalently attached to the 3 acceptor stem of the tRNA by proteins called tRNA synthetases tRNA Gln bound to glutaminyl-tRNA Gln synthetasetRNA cloverleaf diagram

17. Aminoacylation occurs by one of two pathways (class I or class II)

18. The interaction between the tRNA, the appropriate amino acid and the tRNA synthetase is exceptionally important for translational fidelity

19. The structure of tRNA Gln bound to its cognate tRNA synthetase demonstrates one mechanism for specificity

20. Identity elements in tRNAs Size of yellow ball is proportional to the fraction of 20 tRNA acceptor types for which the nucleoside is an observed determinant Diagram of tRNA identity elements

21. tRNA synthetases can edit incorrect aminoacylation events as well

22. There are at least 3 major issues: 1.Proper amino acid must be attached to every tRNA 2.Proper binding of tRNA (anticodon) to mRNA (codon) must occur 3.Triplet code must be interpreted in the proper frame Problem #1 is solved!

23. How do the appropriate tRNAs bind to the correct triplet codon?

24. Codon : Anticodon binding specificity

25. Base pairing rules for the THIRD position of the codon Illustration of non-specific interactions with inosine

26. Codon: 5-CAC-3 Anticodon: 3-GUG-5 Codon: 5-CAU-3 Anticodon: 3-GUG-5 A wobble example

27. There are at least 3 major issues: 1.Proper amino acid must be attached to every tRNA 2.Proper binding of tRNA (anticodon) to mRNA (codon) must occur 3.Triplet code must be interpreted in the proper frame Problem #1 is solved! and Problem #2 is solved

28. How does translation choose the correct reading frame of the triplet code?

29. The reading frame problem, illustrated:

30. In this case, the solution is easy: Specific initiation of translation at a 5 methionyl- tRNA codon (AUG) Strict, 3-nucleotide transitions during translation elongation

31. There are at least 3 major issues: 1.Proper amino acid must be attached to every tRNA 2.Proper binding of tRNA (anticodon) to mRNA (codon) must occur 3.Triplet code must be interpreted in the proper frame All three problems are solved…

32. There are at least 3 major issues: 1.Proper amino acid must be attached to every tRNA 2.Proper binding of tRNA (anticodon) to mRNA (codon) must occur 3.Triplet code must be interpreted in the proper frame All three problems are solved… Now: What molecular machine executes the process of translation?

33. Topics covered in this portion of the lecture (the rest of today and Monday): Prokaryotic ribosome structure Prokaryotic translation Prokaryotic versus Eukaryotic: Ribosome features Translation mechanisms Two examples of medical impact of translation The ribosome and translation

34. The prokaryotic ribosome structure has been solved at atomic resolution The bacterial ribosome is: 2 subunits (50S and 30S) 3 ribosomal RNAs (rRNAs) 52 proteins Total Mass = ~2.5 million Daltons

35. Alberts 6-64d? A low resolution structure to understand organization of sites in the ribosome

36. Alberts 6-64d? A low resolution structure to understand organization of sites in the ribosome How did the field progress from this cartoon to understanding molecular details of this massive cellular machine?

37. Early cryo-electron microscopy experiments revealed the general shape of the ribosome: led to initial nomenclature

38. Better techniques led to better models: Three dimensional model of the 70S ribosome CP, central protuberence SP, spur Cate et al. (1999) Science 285:2097.

39. Better EM models permit visualization of the functional center of the 70S ribosome Liljas (1999) Science 285:2077. Aminoacyl Peptidyl Exit A P E AP E

40. Large (50S) Subunit Proteins-purple 23S rRNA-orange & white 5S rRNA (top)-burgundy & white A site tRNA- green P site tRNA- red From Cech, Science 289: 878 (2000) The crystal structure of the prokaryotic large subunit

41. Large (50S) Subunit Proteins-purple 23S rRNA-orange & white 5S rRNA (top)-burgundy & white A site tRNA- green P site tRNA- red From Cech, Science 289: 878 (2000) No protein sidechain atoms lies within 18 angstroms of the peptidyl transferase site, so ribosome is officially a ribozyme. The crystal structure of the prokaryotic large subunit

42. Schluenzen et al., Cell 102: 615 (2000) Head Platform Body Foot Shoulder Nose The small (30S) subunit: RNA = gold ribbon Proteins = colored ribbons The crystal structure of the prokaryotic small subunit

43. Functional sites mapped onto spacefill model of large and small subunits Green = A site Blue = P site Yellow = E site

44. 2009 Nobel Prize in Chemistry was awarded for structural insights into ribosome function -picture from New York Times From left to right: Venkatraman Ramakrishnan MRC Laboratory of Molecular Biology, Cambridge, United Kingdom Thomas A. Steitz Yale University, New Haven, CT, USA Ada E. Yonath Weizmann Institute of Science, Rehovot, Israel

45. That is the ribosome. Nest question: What is translation and how does it work? We will deal with that on Monday!

46. THE END -any questions?