Nirenberg Cracks the Genetic Code

Marshall W. Nirenberg developed a breakthrough experimental method that unraveled the genetic code, enabling scientists to understand the language in which DNA describes and creates the features of living organisms.

Summary of Event

Nucleic acids somehow carry the blueprint for the building of proteins; this had been confirmed by 1944 and accepted conclusively by 1952. Nevertheless, it took James D. Watson and Francis Crick’s discovery of the molecular structure of deoxyribonucleic acid (DNA), in 1953, to suggest how the necessary processes of information storage, replication, and transmission might occur. Their model served to fix the course of subsequent research that would eventually lead to a deciphering of the genetic code and an understanding of the relation between protein and the genetic material. Genetics;genetic code
Deoxyribonucleic acid;code
Ribonucleic acid
[kw]Nirenberg Cracks the Genetic Code (Summer, 1961)
[kw]Genetic Code, Nirenberg Cracks the (Summer, 1961)
Genetics;genetic code
Deoxyribonucleic acid;code
Ribonucleic acid
[g]North America;Summer, 1961: Nirenberg Cracks the Genetic Code[06980]
[g]United States;Summer, 1961: Nirenberg Cracks the Genetic Code[06980]
[c]Genetics;Summer, 1961: Nirenberg Cracks the Genetic Code[06980]
[c]Science and technology;Summer, 1961: Nirenberg Cracks the Genetic Code[06980]
Nirenberg, Marshall W.
Matthaei, J. H.
Crick, Francis
Watson, James D.
Gamow, George
Grunberg-Manago, Marianne
Marshall, Richard E.

Proteins make up most cellular structures and also serve as catalysts for almost all chemical reactions in living organisms. Proteins are polymers (molecular chains composed of similar chemical units) that are made of amino acids Amino acids , of which there are twenty main types. The linear sequence of amino acids is the primary structure of the protein, and their order causes the molecule to fold into a three-dimensional form. The final shape of the molecule is the most important factor in determining its function; therefore, proteins are direct products of the primary sequence of amino acids.

DNA is also a polymer, but it is composed of four different types of links, called nucleotides, or bases, which are attached to a backbone of alternating phosphate and sugar groups. Two chains usually intertwine together in the characteristic double-helical form so that the bases pair up in a regular fashion. There are four major types of bases, called adenine (A), guanine (G), thymine (T), and cytosine (C); they join by hydrogen bonds so that A’s always pair with T’s, and G’s always pair with C’s. Ribonucleic acid (RNA) has a similar structure, but uracil (U) replaces thymine.

In 1953, George Gamow, a theoretical physicist who had been inspired by the Watson-Crick DNA model, came up with an idea that would define the discussion of the coding problem. Noting the four-base linear structure of DNA and the linear primary structure of proteins, he theorized that the order of the former completely determined the latter, rather like a template, and so reasoned that the problem was to figure out how the four-letter “alphabet” of nucleic acid bases could be formed into “words” that would translate into the twenty-letter alphabet of amino acids.

If the words were one letter long, then four nucleotides could obviously code only for four amino acids. Two-letter sequences could combine to get only sixteen. A three-letter sequence, which could allow sixty-four combinations, called “codons,” was therefore the minimum needed to get the magic twenty amino acids. Together with his statement of the problem, published in Nature in 1954, Gamow proposed an ingenious three-letter solution, the “diamond” code, based upon what he took to be twenty types of diamond-shaped pockets (into which he thought the amino acids could fit) formed by the bases in the double helix. This proposal spurred interest in the coding problem, and a flurry of theoretical work followed.

Gamow’s diamond code, however, ran into immediate difficulties. There were mechanical and chemical problems; for example, the model required that protein production take place in the cell nucleus, whereas evidence suggested that it occurred in the cytoplasm, perhaps mediated in some way by RNA (as was later confirmed). Also, in order to fit the spacing of the diamonds to typical amino acid spacing, the code had to be fully overlapping; that is, each code letter in a chain would be used in three codons in a row. This overlapping structure, however, ruled out certain sequences that were known to exist. A variety of other overlapping codes was suggested, but it was eventually shown to be impossible for a fully overlapping structure to work.

If the code was nonoverlapping and was to be read as a series of triplets, then the problem of punctuation arose. Unless there was something that functioned like commas, there was no way for the cell to know where a triplet began. To avoid this problem, creative attempts were made to devise codes that included “nonsense” triplets (that is, ones that did not correspond to any amino acid), and there was some excitement when mathematical permutations of this approach were discovered that produced codes with exactly the magic number of twenty “sense” combinations.

Marshall W. Nirenberg.

(The Nobel Foundation)

This approach, however, implied that DNA molecules in all species should have more or less the same composition, but as the data increased, it was seen that the ratio of A-T pairs and G-C pairs could vary tremendously. Another approach assumed that the combination of bases in a triplet and not their order was important, but mechanisms that might correspond to such a system seemed physically implausible. The theorists might have continued speculating in this fashion, but numerological speculation was cut off abruptly when the key to the code was provided by an experimentalist.

In the summer of 1961, the Fifth International Congress of Biochemistry Fifth International Congress of Biochemistry (1961) was held in Moscow. Crick was among some six thousand participants, as was Marshall W. Nirenberg, a young unknown researcher from the National Institutes of Health in Bethesda, Maryland. Nirenberg was slated to give a ten-minute talk, and the preprinted abstract gave no hint of the discovery he would report, a discovery that he had made after submitting the abstract. Crick, however, was alerted to the content of the paper and was so impressed that he invited Nirenberg to read it again at a much larger session over which he was to preside. Crick recalls that the audience was “startled” and “electrified” by the report; Nirenberg had shown experimentally that the RNA triplet UUU was a codon for the amino acid phenylalanine; the first word of the genetic code had been translated.

Nirenberg and his associate J. H. Matthaei had discovered how to add an RNA message to a test-tube system that synthesized proteins and how to find out what amino acid was synthesized by it. The technique involved extracting the protein synthesis machinery (ribosomes, messenger RNA, and enzymes) from Escherichia coli, the bacillus that inhabits the human gut. Such extracts, when given an energy source such as adenosine triphosphate (ATP), are called “cell-free systems” and are able to incorporate amino acids into protein. Cell-free systems had been developed by other researchers several years earlier but were unreliable because of rapid disintegration of the enzymes and messenger RNA.

Nirenberg and Matthaei were able to increase stability by adding a chemical that allowed them to freeze the system for storage without loss of activity. The twenty amino acids, radioactively labeled with carbon 14, were added to the system. When these ingredients were mixed, only a very little incorporation of amino acid into protein occurred, as revealed by measurements of radioactivity. Next, the artificial RNA message was added, a sequence consisting entirely of uracil bases. (The process of getting a synthetic RNA of known base sequence had been developed by Marianne Grunberg-Manago. In 1955, Severo Ochoa Ochoa, Severo , following Grunberg-Manago’s discovery, developed the enzyme, polynucleotide phosphorylase, which catalyzes such synthesis.) This produced an eight-hundred-fold increase in the radioactivity level; amino acid had been incorporated into protein. Subsequent tests with different mixtures containing only one radioactive amino acid at a time revealed that it was phenylalanine.


After Nirenberg’s discovery in 1961, work proceeded rapidly. By the following year, Crick’s laboratory had confirmed by genetic studies that the code was indeed a triplet code. Nirenberg, Ochoa, Grunberg-Manago, and other researchers had correctly decoded thirty-five of the triplets by 1963. A refined trinucleotide binding test developed in Nirenberg’s laboratory increased the number to fifty, and by 1966 all but three of the sixty-four possible triplets had been assigned to their corresponding amino acid; most had been reconfirmed in different laboratories by more than one experimental method. The final three triplets, UAA, UAG, and UGA, were revealed to be chain terminators—punctuation that specified the end of an amino acid “sentence”—and this brought the coding problem, as it was originally conceived, to a final solution.

Answers, however, always generate more questions. It had been shown in 1944 by Oswald T. Avery Avery, Oswald T. and his colleagues that DNA was the genetic material among almost all species of organisms, but it was not known if the genetic code was universal. Some of the theoretical arguments that had been made before Nirenberg’s breakthrough had relied upon the assumption of universality, based upon the idea that physical and chemical properties of the twenty amino acids would constrain possible codon assignments. After it was learned that “suppressor” mutations can change a codon reading, universality could no longer be taken for granted. Experimental work on the question, however, has for the most part confirmed the code’s universality.

Richard E. Marshall, C. Thomas Caskey Caskey, C. Thomas , and Nirenberg performed the most extensive research, and their results were published in 1967 in Science. Making use of the trinucleotide-binding technique, they showed that cell-free preparations from a bacterium (E. coli), a mammal (guinea pig), and an amphibian (toad) gave almost identical results for most of the fifty different RNA codons tested. Confirming evidence was quickly obtained in studies using various techniques and a wide variety of organisms and cell types, for example, tobacco mosaic virus, plant coat proteins, rat liver, and human hemoglobins. A very few exceptions have been found, particularly in mitochondria, but these are best regarded as evolutionary deviations from a single standard code.

From a theoretical standpoint, the near universality of the code supports the hypothesis that all forms of life on Earth are related to one another by common evolution; at some point very early in the evolution of life, a single chemistry emerged as adaptively successful and all subsequent variation has built upon that structure. From the standpoint of applied science, the universality of the code simplifies the technology of bioengineering, since it allows bits of DNA from one organism to be spliced into that of another organism. The impact of this technology, which was made possible by building upon the sorts of techniques developed to elucidate the code and its related chemical mechanisms, is only beginning to be felt, and it has given rise to new legal and ethical problems that have yet to be resolved. Genetics;genetic code
Deoxyribonucleic acid;code
Ribonucleic acid

Further Reading

  • Crick, Francis. What Mad Pursuit: A Personal View of Scentific Discovery. New York: Basic Books, 1988. Crick’s intellectual autobiography is a highly readable history of the key events in the investigation of the structure of DNA and the genetic code. Chapters 8 through 12 describe, from Crick’s point of view, the nature of the coding problem and the research conducted on the problem. An appendix gives a clear introduction to the basic molecular mechanisms needed for an understanding of genetics, and a second appendix gives the complete code.
  • Haseltine, William A. “The Genetic Code.” In The Microverse, edited by Byron Preiss and William R. Alschuler. New York: Bantam Books, 1989. Written as an approachable introduction for the layperson, this is a good first source for an overview of the current state of the art in molecular genetics. Of particular interest is the description of advances in understanding of genetic mechanisms that have occurred since the breaking of the code, especially the relevance of genetics to cancer and AIDS research.
  • Kay, Lily E. Who Wrote the Book of Life? A History of the Genetic Code. Stanford, Calif.: Stanford University Press, 2000. History of the breaking of the genetic code that places it as one event in the larger dawning of the information age. Relates DNA to cryptography, linguistics, computer science, and cultural history. Bibliographic references and index.
  • Lappé, Marc. Broken Code: The Exploitation of DNA. San Francisco: Sierra Club Books, 1984. Outlines advances following the breaking of the code that resulted in recombinant DNA (rDNA) technology, but the focus of the book is on the ethical ramifications of genetic engineering as they have been debated in the public and political arenas. The book contains an index, glossary of terms, bibliographical references following each chapter, and useful appendixes.
  • Lewin, Benjamin. Genes III. 3d ed. New York: John Wiley & Sons, 1987. A popular introductory college-level textbook on the molecular biological basis of genetics. Clear explanations are given of the current view on key genetic concepts and biomolecular mechanisms. A complete glossary defines all technical terminology, and there is an exhaustive index.
  • Nirenberg, Marshall W. “The Genetic Code: II.” Scientific American, March, 1963, 80-94. Describes the technique Nirenberg used to match nucleotide triplets to the amino acid they code for. Some material has been superceded. Interesting to compare this article with the ones by Francis Crick that appeared in Scientific American in October, 1962 (“The Genetic Code”) and October, 1966 (“The Genetic Code: III”) to get a sense of how quickly the field developed.
  • Pouplana, Lluís Ribas de, ed. The Genetic Code and the Origin of Life. New York: Kluwer Academic/Plenum, 2004. Compilation of essays on the genetic code and human evolution. Bibliographic references and index.
  • Yčas, M. The Biological Code. Amsterdam: North-Holland, 1969. Although somewhat technical in orientation this book should nevertheless be accessible to a dedicated reader; however, because so much has been learned since it was published, it should not be consulted for timely material. Useful for its explanations of important experimental work and its extensive bibliography. Contains name and subject indexes.

Avery, MacLeod, and McCarty Determine That DNA Carries Hereditary Information

Bevis Describes Amniocentesis as a Method to Check Fetal Genetic Traits

Watson and Crick Announce the Double-Helix Model for DNA

Ochoa Creates Synthetic RNA

Horsfall Detects the Link Between Cancer and Altered DNA

Kornberg and Colleagues Synthesize Biologically Active DNA