Nirenberg and Matthaei Experiment - Experimental Work

Experimental Work

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.