Role of the formylmethionine codon AUG in phasing translation of synthetic messenger RNA

Abstract

The coding specificity of the triplets AUG and UUG was measured in the binding assay of Nirenberg & Leder (1964) , as a function of the magnesium ion and oligonucleotide concentration. AUG was extremely active in promoting the binding of formylmethionine-sRNA under all conditions tested, while UUG was active only at very high magnesium ion and oligonucleotide concentration. These results suggest that AUG is the correct formylmethionine codon. When the AUG codon is incorporated into longer polynucleotide chains, it suppresses the reading of codons which partially overlap its sequence, and promotes the reading of the adjacent 3′ codon. For example, ApUpG ( pU ) 15 ¯ stimulates the binding of Met- and Phe-sRNA, but not of Val-sRNA; in contrast, ApUpGpG ( pU ) 16 ¯ codes for Met- and Val-sRNA, whereas the binding of Phe-sRNA is reduced to a low level, comparable to oligo U. The triplets ACG, UAG and AAG do not show this phasing activity. AUG is capable of fixing the reading frame of the message regardless of its position within the polynucleotide chain. Thus ApApApUpG stimulates strongly the binding of Met-sRNA, but not of Lys-sRNA. On the other hand, ApApApCpG codes very well for Lys-sRNA. The phasing activity of AUG is a maximum at about 0·009 M -Mg 2+ , and decreases sharply at higher concentrations, where the selection of a reading frame is made more or less at random. Since the initiation of polypeptide synthesis directed by natural messengers also occurs best at a low magnesium ion concentration, it is possible that the AUG phaser may be a part of the natural initiation mechanism.