Curiosity is the Key to Discovery

The Life and Scientific Work ofMarshall W. Nirenberg

BTC-575 Scientific Discovery

Presented by Prasanna Khandavilli

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”

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

"for their interpretation of the genetic code and its function in protein synthesis"

 

                                               

Robert W. Holley Har Gobind Khorana Marshall W. Nirenberg

1/3 of the prize 1/3 of the prize 1/3 of the prize

USA USA USA

Cornell University Ithaca, NY, USA

University of Wisconsin Madison, WI, USA

National Institutes of Health Bethesda, MD, USA

b. 1922d. 1993

b. 1922(in Raipur, India)

b. 1927

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)

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

The Genetic Code

Nucleic acid:

• Template for other molecules

• Biological Clock

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

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

The Concept of a Gene-Protein Code

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

Cell-free synthesis of Penicillinase

Pollock : - Molecular weight of Penicillinase is low - The enzyme lacks Cysteine DNAase inhibited in vitro Amino Acid incorporationinto protein

Cell-free Synthesis of Protein dependent upon DNA templates

Heinrich MatthaeiHeinrich MatthaeiRNA-dependent Protein Synthesis RNA-dependent Protein Synthesis

Source: Nobel Lecture Nirenberg

Effect of DNAase & mRNA upon incorporation of Valine into protein in E.coli extracts

RNA from Yeast, Ribosome &Tobacco Mosaic Virus –

Active in Amino Acid incorporation

Single-stranded Poly-U :

An active template for Phenylalanine incorporation

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

RNA-dependent Protein Synthesis

Base Composition of Codons

Cell-free Protein Synthesis with randomly-ordered RNA templates containing different combinations of bases

Polynucleotide Phosphorylase

Table 1:-

Source: Nobel Lecture Nirenberg

Minimum species of bases required for mRNA codons

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

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) ?

Table II

Source: Nobel Lecture Nirenberg

Lys-tRNA binding to ribosomes

Conclusions at this stage: Code is Triplet.

What Next? Codon Base sequence?

(UUG/UGU/GUU?) Triplets are translated in a non-overlapping

fashion ?

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

Fractionation of Poly-(U,G) digests

GUU – Valine

UGU – Cysteine

UUG - Leucine

Base Sequence Studies

Figure 2

Source: Nobel Lecture Nirenberg

Problem: Not able to synthessize all triplet codons enzymatically (not able to proceed further)

Har Gobind Khorana: Synthesis of nucleotides by chemical

methods

Nucleotide Synthesis

Figure 3

Source: Nobel Lecture Nirenberg

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)

Figure 4:PunctuationFigure 4:Punctuation

Source: Nobel Lecture Nirenberg

Figure 5Figure 5

Source: Nobel Lecture Nirenberg

InitiationInitiation

E.Coli

• N-formyl-tRNAf

• 3 non-dializable factors and GTP

• Met-tRNAm- Methionine at internal positions in Protein

• 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!)

Figure 6Figure 6

Data from Rottman and Nirenberg

TerminationTermination

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)

Release of Peptides from Ribosomes:

• Release Factor

• Terminator Codon

Table IIITable III

Source: Nobel Lecture Nirenberg

Codons corresponding to initiation or termination of protein synthesis in E.Coli

• What is the reason for the Degeneracy of the Triplet Code?

• What is its significance?

Source: Nobel Lecture Nirenberg

Codons recognized by species of E.Coli AA-tRNA

RedundancyRedundancy

Synonym Codons:

U equivalent to C

A equivalent to G

Redundant AA-tRNA fractions:

• Products of the same gene

-Altered by enzymes in vivo

-Altered in vitro during fractionation

• Products of different genes

Table VTable V

Source: Nobel Lecture Nirenberg

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

Alternate Base Pairing

• Is the Genetic Code the same?

• With the same DEGENERACY in all organisms?

UniversalityUniversality

• Bacterial, Amphibian, and Mammalian AA- tRNA responses to trinucleotide codons

• Identical Translations of Nucleotide Sequences to Amino Acids

Source: Nobel Lecture Nirenberg

• Mammalian Ile-tRNA : AUU, AUC, and AUA

• E.Coli Ile-tRNA : AUU and AUC

• Mammalian Arg-tRNA : ACG but not AGA

Universality-Conclusions

Reliability of TranslationReliability 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

Each Amino Acid is selectedindependently of other Amino Acids

Errors of Translation are not cumulative

Translation

Precision of Codon Recognition

• Temperature of incubation

• pH

• Concentration of tRNA

• Concentration of Mg2+

• Aliphatic amines Putrescine, Spermidine, Spermine, Streptomycin and related Antibiotics

Rate of TranslationRate 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

FINALLY….

• The Puzzle of the Crick’s Central Dogma of Life Cycle was proved and Explained.

• Genetic Code was deciphered.

• 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

Another Nobel Prize?Another Nobel Prize? at 76 YEARS? at 76 YEARS?

ReferencesReferences• Profiles in Science, National Library of Medicine.

• The Marshall W.Nirenberg papers.

• The Life and Scientific Work of

Marshall W. Nirenberg by Nigel J.T.Thomas PhD CaliforniaStateUniversity, LosAngeles.

• The Nobel Prize Internet Archive.

• Nobel e-Museum Website.

QUESTIONS ?QUESTIONS ?

THANK YOUTHANK YOU