Protein Synthesis 1 Major topics covered: The genetic code

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

2. The Central Dogma of Biology: DNA RNA protein

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

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. “…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: Francis Crick proposed/predicted the Adaptor Hypothesis Crick’s Predictions: 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.

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 • Translation (a collaboration of tRNA and ribosomes) must crackthe 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: Proper amino acid must be attached to everytRNA Proper binding of tRNA (anticodon) to mRNA (codon) must occur 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 tRNAsynthetases tRNA cloverleaf diagram tRNAGln bound to glutaminyl-tRNAGlnsynthetase

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 tRNAsynthetase is exceptionally important for translational fidelity

19. The structure of tRNAGln bound to its cognate tRNAsynthetase demonstrates one mechanism for specificity

20. Diagram of tRNA “identity elements” 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

21. tRNAsynthetases can edit incorrect aminoacylation events as well

22. There are at least 3 major issues: Proper amino acid must be attached to everytRNA Proper binding of tRNA (anticodon) to mRNA (codon) must occur 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. A “wobble” example Codon: 5’-CAC-3’ Anticodon: 3’-GUG-5’ Codon: 5’-CAU-3’ Anticodon: 3’-GUG-5’

27. There are at least 3 major issues: Proper amino acid must be attached to everytRNA Proper binding of tRNA (anticodon) to mRNA (codon) must occur 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- tRNAcodon (AUG) • Strict, 3-nucleotide transitions during translation elongation

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

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

33. The ribosome and translation • 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

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

36. A low resolution “structure” to understand organization of sites in the ribosome Alberts 6-64d? 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 Aminoacyl Peptidyl Exit E P E A P A Liljas (1999) Science 285:2077.

40. The crystal structure of the prokaryotic large subunit • 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)

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

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

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 From left to right: VenkatramanRamakrishnanMRC Laboratory of Molecular Biology, Cambridge, United Kingdom Thomas A. SteitzYale University, New Haven, CT, USA Ada E. YonathWeizmann Institute of Science, Rehovot, Israel -picture from New York Times

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?