preparing synthetic polyribonucleotides of various compositions using a special enzyme, polynucleotide phosphorylase, was first demonstrated by Grunberg-Manago and Ochoa several years earlier (1955). The composition of polynucleotides synthesized in the system that they described depended only on the selection of ribonucleoside diphosphates supplied as substrates; homopolynucleotides such as polyuridylic acid, polyadenylic acid, and polycytidylic acid prepared from UDP, ADP, and CDP, respectively, were the simplest polyribonucleotides synthesized. Using poly(U) as a template polynucleotide for E. coli ribosomes, Nirenberg and Matthaei (1961) demonstrated that this template directs synthesis of polyphenylalanine. It has been concluded that the triplet UUU codes for phenylalanine. Similarly, experiments with polyadenylic and polycytidylic acids have shown that AAA codes for lysine, and CCC for proline.

Further elucidation of the genetic code was based on the use of synthetic statistical heteropolynucleotides of a different composition, which was set by the number and ratio of substrate nucleoside diphosphates in the polynucleotide phosphorylase reaction (Nirenberg et al., 1963; Speyer et al., 1963). Thus, it was demonstrated that the statistical poly(U, C) copolymer directed the incorporation of four amino acids into the polypeptide chain; these were phenylalanine, leucine, serine, and proline. If the U-to-C ratio in the polynucleotide was 1:1, then all four amino acids were incorporated into the polypeptide with equal probabilities. If the U-to-C ratio was 5:1, the probabilities of amino acid incorporation were as follows: Phe > Leu = Ser > Pro. Thus phenylalanine should be coded by triplets consisting of three U or of two U and one C. Leucine and serine are coded by triplets consisting of two U and one C or of two C and one U. Proline is coded by triplets consisting of three C or of two C and one U. Unfortunately, this approach could provide only the composition of the coding triplets, not their nucleotide sequence, since the nucleotide sequence of the template polynucleotide used was statistical.

Due to the invention of a new technique by Nirenberg and Leder (1964), the nucleotide sequences of the coding triplets were soon determined. They found that individual trinucleotides possessed coding properties: after association with the ribosome they supported the selective binding of aminoacyl-tRNA species with the ribosome. For example, UUU and UUC triplets stimulated the binding of phenylalanyl-tRNA, UCU and UCC the binding of seryl-tRNA, CUU and CUC the binding of leucyl-tRNA, and CCU and CCC the binding of prolyl-tRNA. By 1964, methods for synthesizing trinucleotides with the desired sequence were available. In the subsequent two years a wide variety of trinucleotides were tested and, as a result, virtually the whole code was deciphered (Fig. 2.2).

The end of the story was marked by the use of synthetic polynucleotides with a regular nucleotide sequence as templates in the cell-free ribosomal systems of polypeptide synthesis. Methods allowing regular polynucleotides to be synthesized have been developed by Khorana, who has also verified the genetic code by directly using these polynucleotides as templates (Khorana et al., 1966). In complete agreement with the previously established code dictionary, the use of poly(UC)n as a template resulted in the synthesis of a polypeptide consisting of alternating serine and leucine residues, while poly(UG)n directed synthesis of the regular copolymer with alternating valine and cysteine residues. Poly(AAG)n directed the synthesis of three homopolymers: polylysine, polyarginine, and polyglutamic acid.

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