Curiosity is the Key to Discovery

1. Curiosity is the Key to Discovery The Life and Scientific Work ofMarshall W. Nirenberg BTC-575 Scientific Discovery Presented by Prasanna Khandavilli

2. Marshall W.Nirenberg Nobel Prize in Physiology or Medicine 1968 Shared with Robert W.Holley and HarGobindKhorana “Deciphering the Genetic Code and interpretation of its function in Protein Synthesis”

3. The Sveriges Riksbank (Bank of Sweden) instituted the Prize in Economic Sciences in memory of Alfred Nobel, founder of the Nobel Prize.

5. Marshall W.Nirenberg • Born April 10, 1927 in New York • B.S. Zoology and Chemistry, University of Florida at Gainesville (1948) • M.S. Zoology, University of Florida (1952) • PhD Biological Chemistry, University of Michigan at Ann Arbor (1957) • Research Biochemist, NIAMDD; begins Poly-U experiments with Heinrich J.Matthaei (1960-62)

6. Marshall W.Nirenberg • Describes Poly-U experiment at International Congress of Biochemistry in Moscow in August 1961 • Molecular Biology Award, National Academy of Sciences 1962 • Completes Sequencing of RNA “code words” for twenty Amino Acids • Shares Nobel Prize in Medicine or Physiology for deciphering the genetic code with Robert W.Holley and Har Gobind Khorana 1968

7. TheGenetic Code Nucleic acid: • Template for other molecules • Biological Clock

8. The Concept of a Gene-Protein Code Avery, MacLeod, and McCarty : DNA is the genetic material Beadle and Tatum : One gene – One enzyme Brachet and Caspersson : Relation of RNA to Protein Synthesis

9. The Concept of a Gene-Protein Code Caldwell and Hinshelwoods : • RNA – Five kinds of units • The four bases and ribose phosphate • Two adjacent units correspond to one Amino Acid Dounce : • Three adjacent bases in RNA correspond to one Amino Acid • Templates necessary for protein synthesis

10. George Gamow : • Double-strand of DNA has binding sites for AAs Watson and Crick : • Pairing of bases in DNA Hershey : • Fraction of RNA rapidly synthesized & degraded in E.Coli infected with T2 bacteriophage Volkin and Astrachan : • Composition of that RNA fraction resembles phage DNA

11. Cell-free synthesis of Penicillinase Pollock : - Molecular weight of Penicillinase is low - The enzyme lacks Cysteine DNAase inhibited in vitro Amino Acid incorporation into protein Cell-free Synthesis of Protein dependent upon DNA templates

12. Heinrich MatthaeiRNA-dependent Protein Synthesis Source: Nobel Lecture Nirenberg Effect of DNAase & mRNA upon incorporation of Valine into protein in E.coli extracts

13. RNA-dependent Protein Synthesis RNA from Yeast, Ribosome &Tobacco Mosaic Virus – Active in Amino Acid incorporation Single-stranded Poly-U : An active template for Phenylalanine incorporation

14. RNA-dependent Protein Synthesis • RNA is a template for Protein • Poly-U : Phenylalanine in Protein • Translation of mRNA affected by both Primary and Secondary Structures of the RNA • Phe-tRNA is an obligatory intermediate in Polyphenylalanine Synthesis

15. Base Composition of Codons • Cell-free Protein Synthesis with randomly-ordered RNA templates containing different combinations of bases • Polynucleotide Phosphorylase

16. Table 1:- Minimum species of bases required for mRNA codons Source: Nobel Lecture Nirenberg

17. Poly-U Phenylalanine Poly-C Proline Poly-A Lysine Poly-G No template activity Poly-(U,C), Poly-(C,G), and Poly-(A,G): Templates for 2 additional Amino Acids per polynucleotide Poly-(U,A), Poly-(U,G), and Poly-(C,A): Templates for 4 additional Amino Acids per Polynucleotide Poly-(U,G): High degree of Secondary Structure in solution Do not serve as templates for Protein Synthesis

18. Conclusions at this stage: • Code is highly degenerate Still Enigmatic Puzzles! • Is the Code a triplet /Duplex/Tetra? (U/UU/UUU/UUUU?) • Codon Base sequence? (UUG/UGU/GUU?) • Triplets are translated in a non-overlapping fashion (UUUAAA) ?

19. Table II Lys-tRNA binding to ribosomes Source: Nobel Lecture Nirenberg

20. Conclusions at this stage: • Code is Triplet. What Next? • Codon Base sequence? (UUG/UGU/GUU?) • Triplets are translated in a non-overlapping fashion ?

21. Base Sequence of Codons Phe-tRNA attaches to ribosomes in response to poly-U prior to peptide bond formation Trinucleotides function as specific templates for AA-tRNA binding to ribosomes

22. Base Sequence Studies Fractionation of Poly-(U,G) digests GUU – Valine UGU – Cysteine UUG - Leucine

23. Figure 2 Source: Nobel Lecture Nirenberg

24. Nucleotide Synthesis Problem: Not able to synthessize all triplet codons enzymatically (not able to proceed further) Har Gobind Khorana: Synthesis of nucleotides by chemical methods

25. Figure 3 Source: Nobel Lecture Nirenberg

26. Systematic Degeneracy: Replacement of one base by another in DNA does not result in the replacement of one Amino Acid by another in Protein. Many mutations are silent. (Synonymous Substitution)

27. Figure 4:Punctuation Source: Nobel Lecture Nirenberg

28. Figure 5 Source: Nobel Lecture Nirenberg

29. Initiation E.Coli • N-formyl-tRNAf • 3 non-dializable factors and GTP • Met-tRNAm- Methionine at internal positions in Protein

30. Are the DNA templates having only A,C,G,T (and U in mRNA)? • What about the other 21 letters? (all bases are chemical modifications of ring structures!)

31. Figure 6 Data from Rottman and Nirenberg

32. Termination Mutant Bacteriophage T4: • “Sense” codon is converted by mutation to a “nonsense” codon • Nonsense mutations within the gene for the head protein of bacteriophage T4 – chain length of the corresponding polypeptide • UAA, UAG and UGA – Nonsense codons (Termination of Protein Synthesis)

33. Release of Peptides from Ribosomes: • Release Factor • Terminator Codon

34. Table III Codons corresponding to initiation or termination of protein synthesis in E.Coli Source: Nobel Lecture Nirenberg

35. What is the reason for the Degeneracy of the Triplet Code? • What is its significance?

36. Codons recognized by species of E.Coli AA-tRNA Source: Nobel Lecture Nirenberg

37. Redundancy Synonym Codons: U equivalent to C A equivalent to G

38. Redundant AA-tRNA fractions: • Products of the same gene -Altered by enzymes in vivo -Altered in vitro during fractionation • Products of different genes

39. Table V Alternate base-pairing Source: Nobel Lecture Nirenberg

40. Alternate Base Pairing Robert W.Holley: Inosine in the Anticodon pairs alternately with U, C, or A, in the third position of the mRNA Codons

41. Is the Genetic Code the same? • With the same DEGENERACY in all organisms?

42. Universality • Bacterial, Amphibian, and Mammalian AA- tRNA responses to trinucleotide codons • Identical Translations of Nucleotide Sequences to Amino Acids

43. Source: Nobel Lecture Nirenberg

44. Universality-Conclusions • Mammalian Ile-tRNA : AUU, AUC, and AUA • E.Coli Ile-tRNA : AUU and AUC • Mammalian Arg-tRNA : ACG but not AGA

45. Reliability of Translation To synthesize one molecule of Protein with 400 Amino Acid residues 400 AA-tRNA molecules must be selected in the proper sequence Synthesis of corresponding molecule of mRNA, 1206 molecules of ribonucleoside triphosphate must be selected in sequence

46. Translation Each Amino Acid is selected independently of other Amino Acids Errors of Translation are not cumulative

47. Precision of Codon Recognition • Temperature of incubation • pH • Concentration of tRNA • Concentration of Mg2+ • Aliphatic amines Putrescine, Spermidine, Spermine, Streptomycin and related Antibiotics

48. Rate of Translation E.Coli Chromosome: • 3*106 base pairs • 1*106 Amino Acids • 2500-3000 species of Protein • 20-80 mRNA triplets translated per second per ribosome at 370C • 1000-15000 ribosomes per chromosome

49. FINALLY…. • The Puzzle of the Crick’s Central Dogma of Life Cycle was proved and Explained. • Genetic Code was deciphered.

50. Another Nobel Prize? at 76 YEARS? • Chief of Biochemical Genetic Laboratory, N.H.I. Professor of Molecular Cell Biology, University of Maryland, College Park. George Washington University Medical Center. Current Research Work…. • Affects of Morphine on the Nervous System • Neural Cell Receptors using Chick Retina • HomoeBox genes in Drosophila