46 Review TRENDS in Biochemical Sciences Vol.29 No.1 January 2004 Historical review: Deciphering the genetic code – a personal account Marshall Nirenberg Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, National Institutes of Health, 9000 Rockville Pike, MSC – 1654, Building 10, Room 7N-315, Bethesda, MD 20892-1654, USA This is an autobiographical description of the events b-galactosidase in Escherichia coli and that the mechan- that led to the breaking of the genetic code and the sub- ism of protein synthesis was one of the most exciting areas sequent race to decipher the code. The code was deci- in biochemistry. Some of the best biochemists in the world phered in two stages over a five-year period between were working on cell-free protein synthesis, and I had no 1961 and 1966. During the first stage, the base compo- experience with either gene regulation or protein syn- sitions of codons were deciphered by the directing cell- thesis, having previously worked on sugar transport, free protein synthesis with randomly ordered RNA glycogen metabolism and enzyme purification. After preparations. During the second phase, the nucleotide thinking about this for a considerable time, I finally sequences of RNA codons were deciphered by deter- decided to switch fields. My immediate objective was to mining the species of aminoacyl-tRNA that bound to investigate the existence of mRNA by determining ribosomes in response to trinucleotides of known whether cell-free protein synthesis in E. coli extracts sequence. Views on general topics such as how to pick was stimulated by an RNA fraction or by DNA. In the a research problem and competition versus collabor- longer term, my objective was to achieve the cell-free ation also are discussed. synthesis of penicillinase, a small inducible enzyme that I would like to tell you how the genetic code was deciphered from a personal point of view. I came to the National Institutes of Health (NIH) in 1957 as a post-doctoral fellow with Dewitt Stetten, Jr, a wise, highly articulate scientist and administrator, immediately after obtaining a PhD in biochemistry from the University of Michigan in Ann Arbor. The next year, I started work with William Jakoby and, by enrichment culture, I isolated a Pseudomonad that grew on g-butyrolactone and purified three enzymes involved in the catabolism of g-hydroxybutyric acid [1]. There was a weekly seminar in Stetten’s laboratory in which Gordon Tomkins (Figure 1), who worked in a different laboratory, participated. Gordon was brilliant, with a wonderful associative memory and a magnificent sense of humor. His seminars were superb, especially his description of the step-by-step developments in the problem that he intended to discuss. Towards the end of my post-doctoral fellowship, Gordon replaced Herman Kalckar as head of the Section of Metabolic Enzymes and offered me a position as an independent investigator in his laboratory. The other independent investigators in the laboratory were Elizabeth Maxwell and Victor Ginsberg, who were carbohydrate biochemists, and Todd Miles, a nucleic-acid biochemist. It was a wonderful opportunity and I decided then that if I was going to work this hard I might as well have the fun of exploring an important problem. In my opinion, the most exciting work in molecular biology in 1959 were the genetic experiments of Monod Figure 1. Gordon Tompkins. Gordon was brilliant, highly articulate and very funny. He was a charismatic individual who created a stimulating atmosphere and and Jacob on the regulation of the gene that encodes encouraged exploration. In 1958, towards the end of my post-doctoral fellowship at the NIH, he offered me a position as an independent investigator in his Corresponding author: Marshall Nirenberg ([email protected]). laboratory. http://tibs.trends.com 0968-0004/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2003.11.009 Review TRENDS in Biochemical Sciences Vol.29 No.1 January 2004 47 lacks cysteine so that I could explore mechanisms of gene regulation. I thought that in the absence of cysteine the synthesis of penicillinase might proceed, whereas syn- thesis of most other proteins might be reduced. In England, Pollock [2] had shown that penicillinase is inducible in Bacillus cerus and had isolated mutants that differed in the regulation of the penicillinase gene. In 1959, tRNAwas recently discovered but mRNAwas unknown. At that time, the only clues that RNA might function as a template for protein synthesis were a report by Hershey et al. [3], showing that a fraction of RNA is synthesized and degraded rapidly in E. coli infected with T2 bacteriophage, and a paper by Volkin and Astrachan [4], which showed that infection of E. coli by T2 bacteriophage resulted in the rapid turnover of a fraction of RNA that had the base composition of bacteriophage rather than the DNA of E. coli. If mRNA did exist, I thought that it might be contained in ribosomes because amino acids were known to be incorporated into protein on these organelles. I estimated it would take me two years to set up a cell-free system to determine whether RNA or DNA stimulated protein synthesis, which was a pretty accurate estimate. I knew this was a risky problem to work on because Figure 2. Incorporation of 14C—labeled valine into protein in Escherichia coli starting out as an independent investigator you are extracts. Endogenous incorporation of radioactive amino acids into protein in supposed to hit the deck running and prove that you are E. coli extracts was high. However, amino acid incorporation ceased after incu- an effective, productive investigator. One evening I saw bation for ,40 min with DNase I. I found that I could freeze E. coli extracts and thaw them without loss of activity, so I incubated E. coli extracts in the absence of Bruce Ames working in his laboratory. Because I thought radioactive amino acids for 40 min, divided the extracts into small aliquots and he was one of the best young scientists at the NIH I froze them for use later in different experiments. Endogenous incorporation of described my research plan and asked for his evaluation. radioactive amino acids was greatly reduced in such extracts, and addition of mRNA preparations from ribosomes clearly stimulated amino acid incorporation He just looked at me and said ‘It is suicidal’. Although we into protein [16,34,49]. Reproduced from Ref. [16]. both agreed that it was a dangerous project to work on, I thought suicidal was a little extreme. On the one hand I After working on this for about for about a year and a wanted to explore an important problem, on the other I half, Heinrich Matthei came to my laboratory as a post- was afraid of failure, but the wish to explore was much doctoral fellow. Heinrich was a plant physiologist from greater than the fear of failure. Germany who was a post-doctoral fellow at Cornell who As soon as I moved to Gordon’s laboratory I started to wanted to work on protein synthesis. He came under the make cell-free extracts that incorporated amino acids into impression that, because the NIH is such a big institution, protein, and to prepare DNA and RNA from ribosomes of many people would be working on protein synthesis. He penicillinase inducible and constitutive strains of B. cerus. stopped in Roy Vagelos’s laboratory and Roy sent him to me I devised a sensitive assay for penicillinase and starting because I was the only person at the NIH who was with conditions that had been devised by Lamborg and studying cell-free protein synthesis. We needed a more Zamecnik and his colleagues [5], I tried to obtain the sensitive assay, so I suggested that Heinrich use the cell- de novo synthesis of penicillinase following addition of free amino-acid-incorporating system that I had optimized either RNA or DNA fractions from either B. cerus or E. coli. to measure the incorporation of radioactive amino acids Systematically, I explored the optimum conditions for cell- into protein. Heinrich insisted on preparing 20 14C-labeled free synthesis and showed that RNA prepared from amino acids by growing algae in the presence of ribosomes of B. cerus that expressed penicillinase con- 14C-bicarbonate, hydrolyzing the protein and purifying stitutively stimulated penicillinase synthesis by 10–15%, each of the 14C-labeled amino acids, because this is what he but RNA from either uninduced ribosomes or DNA had no had done previously. effect. However, the stimulation of penicillinase synthesis Using this more sensitive assay it was immediately was small and it was clear that I needed a more sensitive apparent that RNA from ribosomes, but not DNA, assay. stimulated incorporation of radioactive amino acids into Usually around noon, Gordon Tomkins would come into protein [6,7]. I jumped for joy because this was the first my laboratory with a sandwich and we would go into the definitive demonstration in vitro that mRNA existed and hall and talk about my work, his work, and various was required for protein synthesis. We fractionated RNA exciting results that had been published. I always stopped from ribosomes and found, as expected, that only a small to talk to him, even though the extract that I was portion stimulated amino acid incorporation into protein [8]. preparing was slowly dying in an ice bucket, because We made three trivial technical advances that had a these were wonderful conversations. Gordon encouraged tremendous effect on our work. First, I established me and created an exciting atmosphere for young conditions that enabled us to freeze and thaw E. coli investigators. extracts with little or no loss in the ability to incorporate http://tibs.trends.com 48 Review TRENDS in Biochemical Sciences Vol.29 No.1 January 2004 him our results.