DISCOVERY OF THE GENETIC CODE – the classic experiment of cell-free protein synthesis

This year, we celebrate the 63rd anniversary of the discovery of the genetic code, the universal cipher of life common to all organisms. The discovery of how the information in DNA is translated to RNA and protein had a tremendous impact on the development of biology, biotechnology, and molecular medicine.

In 1961, Marshall Warren Nierenberg and Johann Heinrich Matthaei developed an experimental system that allowed them to uncover and understand the genetic code. On May 27, 1961, at three o’clock in the morning, they conducted a groundbreaking experiment that showed that polyuridylic RNA (poly-U) encodes phenylalanine, thus proving that RNA can stimulate protein synthesis. The scientists used carbon-labeled phenylalanine in an Escherichia coli extract, which confirmed that the genetic code consists of three nucleotides, and the triplet UUU codes for phenylalanine during protein synthesis on the ribosome. In a similar manner (using a poly-C template), they obtained polyproline and demonstrated that the triplet CCC codes for proline. A key step in the discovery of the genetic code was precipitating homopolypeptides in trichloroacetic acid (TCA). Polyphenylalanine and polyproline, which do not dissolve in TCA, precipitated onto filters. In subsequent studies, Nirenberg and Matthaei also demonstrated that RNA must be single-stranded to act as mRNA, which was crucial for understanding the genetic code and its role in protein synthesis.

By 1966, the entire genetic code was deciphered. Marshall Nirenberg and his team completed defining the genetic code, enabling its graphical representation, which allowed for the identification of amino acids based on nucleotide sequences. Har Gobind Khorana confirmed and expanded the concept of the genetic code, demonstrating that nucleic acids direct the incorporation of amino acids into proteins. Robert William Holley conducted research on the chemical structure of transfer ribonucleic acid (tRNA). In his laboratory in 1965, the first alanine tRNA (tRNAAla) was sequenced.

It was identified that there are 61 codons that encode 20 different amino acids, three stop codons, and the AUG codon that can be used as a start codon. It was discovered that the genetic code is universal for all organisms. It was shown that the genetic code is unambiguous (each codon codes for one amino acid) and degenerate (one amino acid can be coded by more than one codon).

In 1968, Marshall Nirenberg, Robert Holley, and Har Khorana were awarded the Nobel Prize in Physiology and Medicine for deciphering the genetic code and discovering its role in protein synthesis.

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