Nirenberg and Matthaei experiment
The Nirenberg and Matthaei experiment was a scientific experiment performed on May 15, 1961, by Marshall W. Nirenberg and his post doctoral fellow, Heinrich J. Matthaei. The experiment cracked the genetic code by using nucleic acid homopolymers to translate specific amino acids.
In the experiment, an extract from bacterial cells that could make protein even when no intact living cells were present was prepared. Adding an artificial form of RNA, poly-U, to this extract caused it to make a protein composed entirely of the amino acid phenylalanine. This experiment cracked the first codon of the genetic code and showed that RNA controlled the production of specific types of protein.
Oswald Avery discovered that the substance responsible for producing inheritable change in the disease-causing bacteria was neither a protein nor a lipid, rather deoxyribonucleic acid (DNA). He and his colleagues Colin MacLeod and Maclyn McCarty suggested that DNA was responsible for transferring genetic information. Later, Erwin Chargaff discovered that the makeup of DNA differs from one species to another. These experiments helped pave the way for the discovery of the structure of DNA. In 1953, with the help of Maurice Wilkins and Rosalind Franklin’s X-ray crystallography, James Watson and Francis Crick proposed DNA is structured as a double helix.
In the 1960s, one main DNA mystery scientists needed to figure out was the number of bases found in each code word, or codon, during transcription. Scientists knew there was a total of four bases (guanine, cytosine, adenine, and thymine). They also knew that were 20 known amino acids. George Gamow suggested that the genetic code was made of three nucleotides per amino acid. He reasoned that because there are 20 amino acids and only four bases, the coding units could not be single (4 combinations) or pairs (only 16 combinations). Rather, he thought triplets (64 possible combinations) were the coding unit of the genetic code. However, he proposed that the triplets were overlapping and non-degenerate.
Seymour Benzer in the late 1950s had developed an assay using phage mutations which provided the first detailed linearly structured map of a genetic region. Crick felt he could use mutagenesis and genetic recombination phage to further delineate the nature of the genetic code. In the Crick, Brenner et al. experiment, using these phages, the triplet nature of the genetic code was confirmed. They used frameshift mutations and a process called reversions, to add and delete various numbers of nucleotides. When a nucleotide triplet was added or deleted to the DNA sequence the encoded protein was minimally affected. Thus, they concluded that the genetic code is a triplet code because it did not cause a frameshift in the reading frame. They correctly concluded that the code is degenerate (triplets are not overlapping) and that each nucleotide sequence is read from a specific starting point.
Marshall Nirenberg and Johann Matthaei both longed to understand how information gets transmitted from DNA to protein. At this time there was a race to crack the code of the DNA language. At the same time, Severo Ochoa was busy working on the coding problem with the help of Leon Heppel, a skillful biochemist capable of making artificial RNAs of defined compositions. Ochoa had a big staff, and Nirenberg was worried he would not be able to keep up. Many NIH scientists helped Nirenberg in deciphering the mRNA codons for amino acids.Nirenberg and his post doctoral fellow Matthaei started their experiments in a lab in Germany and completed them in a National Institutes of Health (NIH) laboratory campus in Maryland.
In order to decipher this biological mystery, Nirenberg and Matthaei needed a cell-free system that would build amino acids into proteins. Following the work of Alfred Tissieres and after a few failed attempts, they created a stable system by rupturing E. coli bacteria cells and releasing the contents of the cytoplasm. This allowed them to synthesize protein, but only when the correct kind of RNA was added, allowing Nirenberg and Matthaei to control the experiment. They created synthetic RNA molecules outside the bacterium and introduced this RNA to the E. coli system. The experiment used 20 test tubes, each filled with a different amino acid. For each individual experiment, 19 test tubes were "cold", and one was radioactively tagged with 14C so they could detect the tagged amino acid later. They varied the "hot" amino acid in each round of the experiment, seeking to determine which amino acid would be incorporated into a protein following the addition of a particular type of synthetic RNA. In their experiments in late May 1961 they had narrowed down the amino acids encoded by Poly-U to Phenylalanine or Tyrosine.
At 3 am on May 27 Matthaei used phenylalanine for the "hot" test tube. After an hour, the control tubes showed a background level of 70 counts, whereas the hot tube showed 38,000 counts per milligram of protein. The experiment showed that a chain of the repeated uracil bases produced a protein chain made of one repeating amino acid, phenylalanine. Therefore, polyU coded for polyphenylalanine, consistent with UUU coding for phenylalanine. At the time the number of bases per codon could not be determined. The two kept their breakthrough a secret from the larger scientific community until they could complete further experiments with other strands of synthetic RNA (such as Poly-A) and prepare papers for publication. Using the three-letter poly-U experiment as a model, the research team discovered that AAA (three adenosines) was the code word or "codon" for the amino acid lysine, and CCC (three cytosines) was the code word for proline. They also discovered that by replacing one or two units of a triplet with other nucleotides, they could direct the production of other amino acids. They found, for example, that a synthetic RNA GUU codes for a valine be added to a developing amino acid chain.
Reception and Legacy
In August, at the International Congress of Biochemistry in Moscow, Nirenberg presented his paper. The experimentation with synthetic RNA in a cell-free system was a key technical innovation. In 1961, when they announced their methods for decoding the relationship of mRNA to amino acids, there was still a lot of experimentation required before the entire code was deciphered. The scientists had to determine which bases made up each codon, then determine the sequence of bases in the codons. This proved to be a tremendous amount of work.
In 1964 and 1965, Nirenberg's postdoctoral researcher, Philip Leder, developed a filtration machine that allowed the NIH research team determine the order of the nucleotides in the codons. This development sped up the process of assigning code words to amino acids. By 1966, Nirenberg announced that he had deciphered the sixty-four RNA codons for all twenty amino acids.
For his ground-breaking work on the genetic code, Nirenberg was awarded the 1968 Nobel Prize in Physiology or Medicine. He shared the award with Har Gobind Khorana and Robert W. Holley. Working independently, Khorana had mastered the synthesis of nucleic acids, and Holley had discovered the exact chemical structure of transfer-RNA.
The New York Times reported on Nirenberg's discovery by explaining that "the science of biology has reached a new frontier," leading to "a revolution far greater in its potential significance than the atomic or hydrogen bomb." Most of the scientific community saw these experiments as highly important and beneficial. However, there were some who were concerned with the new area of Molecular Genetics. For example, Arne Wilhelm Kaurin Tiselius, the 1948 Nobel Laureate in Chemistry, asserted that knowledge of the genetic code could "lead to methods of tampering with life, of creating new diseases, of controlling minds, of influencing heredity, even perhaps in certain desired directions."
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Deciphering the Genetic Code: Marshall Nirenberg - Office of NIH History