Presenters: Michelle Connors and Ken Fullerton

Note: We were unable to find Nirenberg and Matthaei's 1961 paper but we did find a 1963 paper that Nirenberg wrote about his experiments in 1961 and some of his follow-up experiments.
Susbstitute article: Nirenberg (1963) "The Genetic Code: II"  Scientific American (March) vol. 190: 80-94.

• Born in New York City on April 10, 1927 and moved to Florida ten years later.
• Originally a zoologist and botanist while enrolled at the University of Florida, Gainesville.
• Later introduced to biochemistry (little studied at the time) working in the Nutrition Laboratory where he learned the technique of using radioactive isotopes to follow the pathways of biochemical reactions.
• B.Sc. (1948) @ University of Florida, M.S. (1952) @ U.F., Ph.D. (1957) @ University of Michigan.
• Appointed Research Biochemist at the National Institutes of Health
• Within a year, meets Johann Matthaei and they begin collaborating on protein synthesis experiments.
• Nobel Prize in 1968

• "Martha Chase effect"
• Other than the fact that he worked on this project no other information could be found on him.

Beadle and Tatum:

• Established the one gene - one enzyme hypothesis

• Structure of DNA determined in mid-1950s along with James Watson.
• First realised that any code using only 2 bases at a time would not have the capacity to specify all of the amino acids found in proteins, yet he thought that 3 bases at a time had too high a capacity.
• If the code uses 2 bases at a time and there are 4 possible nucleotides,

then we get 4² = 16 amino acids / code words
• Using 3 bases at a time with 4 nucleotides, we get 4³ = 64 amino acids / code words
• Was not known that there were 20 amino acids.
• Worked with Sidney Brenner and colleagues and established the standard 20 amino acids.
• Knowing there were 64 possible code words and now 20 amino acids suggested that the genetic code may be degenerate.

• Coined the term messenger RNA (mRNA) to describe the template RNA that carried genetic messages from the DNA to the ribosomes, although mRNA was not yet isolated at the time of Nirenberg and Matthaei's experiment.
• Enzymatic synthesis of RNA complementary to DNA proven shortly thereafter by Hurwitz, Weiss, and Stevens
• Enzyme = RNA polymerase

Along with mRNA, it was thought that another form of RNA acted as an "adaptor molecule" to bring the amino acids to the proper sites on the mRNA.  This "adaptor molecule" was the transfer RNA (tRNA).


"Although direct recognition of messenger RNA code words by transfer RNA molecules has not been demonstrated, it is clear that these molecules preform at least part of the job of placing amino acids in the proper position in the protein chain." - Nirenberg (1963)
 

• At the time, the tRNA was thought to be a special helical form of RNA transporting the amino acids.  At least one tRNA for each one, but all seem to carry the bases ACC at the point of amino acid attachment and G at the opposite end.  See Figure 1.

Experimental Setup/Methods
 

• The basic idea of this experiment is best summarized by Nirenberg himself:

 

 

"If one could establish the base sequence in one of the cell's genes - or part of a gene - and correlate it with the amino acid sequence in the protein coded by that gene, one would be able to translate the genetic code"   - Nirenberg (1963)
 

• Experiment drew on methods devised by Paul Zamecnik a few years earlier.  Zamecnik had developed a cell-free in vitro translation system which was capable of directing the synthesis of radioactively labelled protein.  This system consisted of a membrane-free cell supernatant, ATP, GTP, radioactively labelled amino acids, and RNA.
• The cell-free suspension was obtained by gently braking E. coli cells by grinding them with finely powdered alumina.  Cell "sap" remains.
• "Sap"= DNA, mRNA, ribosomes, cellular enzymes, and Adenosine Triphosphate (ATP).  ATP should provide adequate energy for the incorporation of amino acids into protein.
• Found new way to stablize sap so that it could be stored for weeks without loss of activity.  No mention, however, of how exactly they did this.  (Freezing possibly?)
• For the experimental RNA template they used Tobacco Mosaic Virus (TMV), see diagram
• As a control RNA template, they used the homopolymer poly(U). Because mRNA was yet to be isolated, the control was synthesized from UDP using a specialized enzyme.
• Specialized enzyme = polynucleotide phosphorylase.
• Enzyme normally favours the degradation of RNA into ribonucleotide diphosphates.  However, the reaction can be "forced" in the opposite direction to favour synthesis of RNA in vitro  using high concentrations of ribonucleotide diphosphates. See Figure 12.2 (K&C).
• Polynucleotide phosphorylase acts in an opposite manner than RNA polymerase in that it does not require a DNA template.  Therefore, addition of a ribonucleotide is random, depending on the relative concentration of the four ribonucleotide diphosphates added to the reaction mixtures.

 

 

Key Point: "The probability of insertion of a specific ribonucleotide is proportional to the availability of     that molecule, relative to other ribonucleotides."  (Klug and Cummings, 1997)
 

• Deoxyribonuclease,or DNase, was used to destroy the DNA in the cell sap.
• Synthetic RNA is added to the DNA-less sap along with all 20 amino acids attached to the appropriate tRNA.
• One amino acid is radioactively labeled with carbon 14, other 19 are normal.
• Mixture incubated and then the protein is extracted and the radioactivity is measured.
• They did not actually expect to get any protein synthesis from the poly(U) template.
• Thankfully, they were wrong!  Results discussed shortly.
• In their first experiment, phenylalanine was the radio-labeled amino acid.
• What the setup looked like:
• See Figure 2, Figure 3, Figure 4, and Figure 5.

Results of Experiment

Incorporation of ¹⁴C-Phenylalanine into Protein:
 

Artificial mRNA	Radioactivity (counts/min)
None	44
Poly U	39,800
Poly A	50
Poly C	38

(After Klug and Cummings, 1997)

• poly U produced a protein chain of Phenylalanine.
• Also used poly C & poly A producing Proline and Lysine, respectively.
• poly G folded in on itself and did not produce a protein.
• Later, tests done with RNA heteropolymers such as poly-AC, poly-AG, poly-AU, poly-CG, poly-CU and poly-GU copolymers.
• For every 1.5 uracils in the poly-U mRNA, one Phenylalanine will be incorporated into a protein
• Idea that maybe the mRNA is "recycled" instead of just being read once and then degraded.
• See table.

Conclusions/Interpretations
 

• Poly-U codes for the amino acid Phenylalanine.
• Poly-C codes for the amino acid  Proline.
• Poly-A codes for the amino acid Lysine.
• mRNA is used more than once.
• Strong evidence that the code was indeed triplet in nature, though not proven absolutely.
• Two main factors that probably play a role in the activity of the mRNA are the length of RNA chain and it's overall structure.
• Based on Crick's hypothesis, code is likely degenerate as well.  Proven later by Khorana's work.
• Hypothesis that though degenerate, the code is likely unambiguous:
• One amino acid ---> many code words ....BUT
• Each code word---> one amino acid.

Applications to Modern Genetics Research
 

• Process opened the door for the complete cracking of the genetic code.

"The Moscow meeting was made especially interesting because of the results reported by Marshall Nirenberg, then almost unknown.  I had heard rumours of these experiments but no details...... I later claimed that the audience was "startled" (I think I originally wrote "electrified") to receive this news.  Seymour Benzer countered this with a photograph showing everyone looking extremely bored!  Nevertheless, it was an epoch-making discovery, after which there was no looking back"
 - from What Mad Pursuit, Francis Crick

• Lead to the conclusion that the genetic code is universal (almost!)
• Determining the genetic basis of certain diseases:
• If you know that a certain enzyme is defective or missing, possible to determine which amino acid is affected and in turn, where the genetic problem is.  (Mutation, Insertion, or Deletion of particular nucleotide)

For further information Contact Either:

Michelle Connors Or Ken Fullerton