Transcript Curiosity is the Key to Discovery

Curiosity is the Key to Discovery
The Life and Scientific Work of
Marshall 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”
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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. 1922
d. 1993
b. 1922
(in Raipur, India)
b. 1927
Marshall W.Nirenberg
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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
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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
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
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 incorporation
into protein
Cell-free Synthesis of Protein dependent upon DNA
templates
Heinrich Matthaei
RNA-dependent Protein Synthesis
Source: Nobel Lecture Nirenberg
Effect of DNAase & mRNA upon incorporation of Valine into protein in E.coli extracts
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
RNA-dependent Protein Synthesis
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RNA is a template for Protein
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Poly-U : Phenylalanine in Protein
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Translation of mRNA affected by both Primary and
Secondary Structures of the RNA
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Phe-tRNA is an obligatory intermediate in
Polyphenylalanine Synthesis
Base Composition of Codons
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Cell-free Protein Synthesis with randomlyordered RNA templates containing different
combinations of bases
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Polynucleotide Phosphorylase
Table 1:Minimum species of bases required for mRNA codons
Source: Nobel Lecture Nirenberg
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:
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Code is highly degenerate
Still Enigmatic Puzzles!
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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
Lys-tRNA binding to ribosomes
Source: Nobel Lecture Nirenberg
Conclusions at this stage:
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Code is Triplet.
What Next?
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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
Base Sequence Studies
Fractionation of Poly-(U,G) digests
GUU – Valine
UGU – Cysteine
UUG - Leucine
Figure 2
Source: Nobel Lecture Nirenberg
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
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:Punctuation
Source: Nobel Lecture Nirenberg
Figure 5
Source: Nobel Lecture Nirenberg
Initiation
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 6
Data from Rottman and Nirenberg
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)
Release of Peptides from Ribosomes:
• Release Factor
• Terminator Codon
Table III
Codons corresponding to initiation or termination of protein synthesis in E.Coli
Source: Nobel Lecture Nirenberg
• What is the reason for the
Degeneracy of the Triplet Code?
• What is its significance?
Codons recognized by species of E.Coli AA-tRNA
Source: Nobel Lecture Nirenberg
Redundancy
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 V
Alternate base-pairing
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
• Is the Genetic Code the same?
• With the same DEGENERACY in all
organisms?
Universality
• Bacterial, Amphibian, and Mammalian AAtRNA responses to trinucleotide codons
• Identical Translations of Nucleotide Sequences
to Amino Acids
Source: Nobel Lecture Nirenberg
Universality-Conclusions
• Mammalian Ile-tRNA : AUU, AUC, and AUA
• E.Coli Ile-tRNA : AUU and AUC
• Mammalian Arg-tRNA : ACG but not AGA
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
Translation
Each Amino Acid is selected
independently of other Amino Acids
Errors of Translation are not cumulative
Precision of Codon Recognition
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Temperature of incubation
pH
Concentration of tRNA
Concentration of Mg2+
Aliphatic amines Putrescine, Spermidine,
Spermine, Streptomycin and related Antibiotics
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
FINALLY….
• The Puzzle of the Crick’s Central Dogma of
Life Cycle was proved and Explained.
• Genetic Code was deciphered.
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
References
• 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 ?
THANK YOU