By the early 1950s, scientists knew that genes were made of deoxyribonucleic acid (DNA) and that specific proteins were the products of specific genes. The exact link between DNA and proteins was less well understood, however. Since proteins are considered the language of life, researchers believed that the DNA molecule, with its four nitrogenous bases, might be the code for this language. This is how the term genetic code originated.
Protein molecules are comprised of amino acids. There are twenty biologically important amino acids. Only four different bases--adenine (A), thymine (T), cytosine (C), and guanine (G)--are found in DNA. When each of these bases combines with a sugar and a phosphate molecule, a nucleotide unit is formed. How could only four different nucleotides code for twenty different amino acids? Scientists reasoned that if a single nucleotide coded one amino acid, only four amino acids could be provided for. If two nucleotides specified one amino acid, then there could be a maximum number of sixteen possible arrangements. George Gamow, a Noble Prize winner, demonstrated that at least three nucleotides in sequence were required to code for a single amino acid. This would provide for sixty-four possible combinations or codons--more than enough different "messages" to code for the twenty amino acids. Marshall Nirenberg, Har Khorana, and Robert Holley, three American biochemists, immediately adopted this three-nucleotide, or triplet codon, hypothesis as a foundation for their research. Each worked independently to discover how DNA influences protein synthesis. It was important to understand how the genetic material in a living cell directs the synthesis of proteins, because these proteins determine the structure and activities of the cell.
Marshall Nirenberg was born in New York City on April 10, 1927. He obtained his Ph.D. from the University of Michigan in 1957. He conducted research as head of the Laboratory of Biochemical Genetics at the National Institutes of Health; there he started his work with the genetic code. He wanted to solve the code by answering the following questions: 1) What was the ratio of the four bases in each triplet that coded for a particular amino acid? 2) What was the exact sequence of the bases in each triplet? and 3) Which triplet coded for which amino acid? To simplify the task of identifying the triplet responsible for each amino acid, he used a man-made ribonucleic acid (RNA) polymer. Chemically, RNA is very similar to DNA, except that RNA is single-stranded and nonhelical and contains the base uracil (U) instead of thymine (T). Nirenberg first used a pure RNA polymer that contained only uracil (U). He found that only one amino acid, phenylalanine, was produced from this code, concluding that the code for phenylalanine must be the triplet UUU. Similarly, a pure cytosine (C) RNA polymer produced only the amino acid proline. Thus, the corresponding code must be CCC.
It wasn't long before a working dictionary of the RNA codes was established. Nirenberg began to notice that certain amino acids could be specified by more than one triplet. Statistical tests were later used to determine the amino acid produced from RNA made of a mixture of U and C by measuring the relative proportions of the resulting proteins. It was found that the code was redundant. A particular amino acid could be specified by more than one codon. Thus, the amino acid serine could be produced from any one of the combinations UCU, UCC, UCA, or UCG. Some triplets didn't specify any amino acid. These "nonsense" triplets signaled the beginning or end of synthesis.
After Nirenberg decoded fifty triplets, he worked on finding the exact orders of the three bases of the triplet. By painstakingly labeling one amino acid at a time with radioactive carbon-14, he passed the experimental material through a filter that retained only the cell's ribosomes and attached amino acids. The amount of radioactivity in the filter was measured to determine exactly which triplet code specified which amino acid. For example, AAC coded for asparagine, while CAA coded for glutamine. He obtained clear results for over sixty of the possible codons using this technique.
Har Khorana used this information to introduce new techniques of comparing DNA possessing a known structure with the RNA it would produce. Khorana was born in 1922 in Raipur, a village in Punjab, which is now part of West Pakistan. He studied for several years at universities throughout the world, joining the faculty at the University of Wisconsin, where he became interested in the genetic code, in 1960. He showed that separate nucleotide triplets do not overlap. He also helped determine the genetic code by re-creating each of the sixty-four possible triplets of DNA nucleotides that work in combinations as instructions for the protein-synthesizing ribosomes within the cell. His work proved that the genetic code is linear and consecutive and confirmed that three nucleotides make up one amino acid.
Khorana and his colleagues also determined the direction in which the code is read. In 1968, Khorana produced the first complete and functional synthetic gene. This achievement proved that it was possible to change genes and observe the results of those changes. This has subsequently been used as a valuable tool to study genetic disorders and the mechanisms of cancerous cells. Artificial genes are now used to obtain large amounts of valuable proteins for human dietary and medical needs.
Robert Holley was born in Urbana, Illinois, on January 28, 1922. He received his Ph.D. from Cornell University in 1947 and became interested in the mechanics of proteins after working with penicillin in World War II. He discovered that small RNA molecules called transfer RNA (tRNA) existed and acted as acceptors for activated amino acids. Holley isolated specific tRNAs for three amino acids--alanine, tyrosine, and valine--in 1958.
As a direct result of Marshall Nirenberg, Har Khorana, and Robert Holley's work, the genetic code has been solved. The twenty amino acids are coded by sixty-one triplet codons; three additional codons do not code for any amino acids but direct the cell as to when it should cease protein synthesis. Each of these chemists shared the 1968 Nobel Prize in Physiology and Medicine for their achievements. Holley died in 1993.
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