By 1960 it was widely recognized that DNA was transcribed to yield messenger RNA (mRNA) and mRNA was translated to yield proteins. The translation step could be carried out in vitro by using extracts from E. coli cells that had been primed with purified RNA. Some of the favorite messages were RNAs from viruses such as TMV or yeast cells. By measuring the amino acids that were incorporated into protein it was possible to show that the RNAs from different sources were making different proteins.
The relationship between the RNA and the protein product was obvious. There was something about the sequence of nucleotides in the RNA molecule that determined the sequence of amino acids in the protein. The RNA encoded the amino acid sequence. What was this genetic code?
Marshall Nirenberg was a scientist working at the NIH labs on the outskirts of Washington D.C. He and his post-doc Heinrich Matthaei realized that they could program their cell free extracts with synthetic RNAs and crack the genetic code.
Their first attempt was with a synthetic RNA called polyU because it was composed entirely of uridine residues [Monday's Molecule #28]. They added polyU to various test tubes containing one amino acid that was radioactively labelled. They then looked for incorporation of this labelled amino acid into high molecular weight protein that could be precipitated from the extract.
Here's how the experiment is described on the NIH website [The poly-U Experiment]
On Saturday, May 27, 1961, at three o'clock in the morning, Matthaei combined the synthetic RNA made only of uracil (called poly-U) with cell sap derived from E. coli bacteria and added it to each of 20 test tubes. This time the “hot” test tube was phenylalanine. The results were spectacular and simple at the same time: 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 repeating bases uracil forced a protein chain made of one repeating amino acid, phenylalanine. The code could be broken! UUU=Phenylalaline was a breakthrough experiment result for Nirenberg and Matthaei.Shortly after discovering the very first codon, Matthaei returned to Germany. Nirenberg assembled a team of scientists to crack the rest of the genetic code by adding various synthetic RNAs to the cell free extract. The first few codons were simple; polyA stimulated incorporation of lysine and polyC stimulated incorporation of proline. (PolyG didn't work.) A random mixture of U and C, poly(U,C), incorporated leucine and serine.
The two kept their breakthrough a secret from the larger scientific community–though many NIH colleagues knew of it –until they could complete further experiments with other strands of synthetic RNA (Poly-A, for example) and prepare papers for publication. They had solved with an experiment what others had been unable to solve with theoretical explanations and mathematical models.
Eventually, with the help of Gobind Korhana, the team was able to synthesize RNAs with defined triplets of nucleotides and the entire genetic code was worked out. Nirenberg, Korhana, and Robert Holley (for determining the structure of tRNA) received the Nobel Prize in 1968.
It's important to note that the cracking of the genetic code for E. coli proved to be universal (almost). All species use the same genetic code. It's also important to note that cracking the genetic code, which was done forty years ago, is not the same as sequencing a genome ["Cracking the genetic code" and "mapping the genome"].
The standard genetic code is shown below. The column on the left represents the first nucleotide in a codon, the row on top (2nd position) represents the middle nucleotide, and the column on the right is the third position. You can see why poly(U,C) encoded serine and leucine because one of the codons for serine is UCU and one of the leucine codons is CUC.