Direction of reading of the genetic message.

The assembly of polypeptide chains during protein biosynthesis is believed to proceed from the NHz-terminal through the COOH-terminal amino acid (l-4). Hence, the most direct method for ascertaining the direction in which the genetic message is read is to determine the end location of a given amino acid in polypeptide chains synthesized in a cell-free system under the direction of synthetic polynucleotides having a codon of specified base sequence at one end of the chain. Previous experiments (5) were inconclusive because of (a) presence of nucleases in the system, (b) insufficient characterization of the polynucleotide messenger, and (c) difficulty of performing end group assays because of the insolubility of the phenylalanine peptides formed. All of these obstacles have now been removed through (a) the use of a system low in nuclease activity consisting of purified Escherichia coli ribosomes and Lactobacillus arabinosus supernatant and (b) the preparation and unequivocal characterization of short polyadenylic acid messengers with 1 cytidine residue (and therefore an AAC codon) at the 3’.end. Lysine polypeptides are soluble in water and can be readily characterized. Experiments to be reported in this paper have shown that polynucleotides of the structure ApApAp . . . pApApC (Ap,C),i with 21 to 23 nucleotide residues, directed the synthesis of a family of asparagine-containing peptides of increasing chain length. The most abundant of these had 1 asparagine per 3 to 5 lysine residues, with NHz-terminal lysine and COOH-terminal asparagine. Hence, if the polypeptide is indeed assembled from the NH2to the COOH-terminal end, the polynucleotide message must be read in the direction from the 5’to the 3’-end or from left to right in the currently accepted fashion of writing poly-