THE IMMEDIATE DOWNSTREAM CODON STRONGLY INFLUENCES THE EFFICIENCY OF UTILIZATION OF EUKARYOTIC TRANSLATION INITIATION CODONS

Nucleotide substitutions were introduced into the initiation site of a n influenza virus NS cDNA derivative at the +4, +5 and +6 positions (w here the A of the AUG codon is defined as +1), in the background of ei ther AUG or CUG as the initiation codon. Capped transcripts of these c onstructs were translated in rabbit reticulocyte lysate under conditio ns where the selection of initiation sites conformed to the scanning r ibosome model. With CUG as the initiation codon, the efficiency of ini tiation was as strongly influenced by the nature of the residue in the +5 position as at +4, whilst the influence of the +6 position was sma ller, The residues favourable to initiation were as follows: at +4, on ly G was stimulatory; at +5, A was strongly stimulatory acid C fairly beneficial; and at +6, only U exerted any positive influence. The posi tive influence of the favourable residues (or the negative influence o f unfavourable residues) at each position appeared to be additive. Wit h AUG as the initiation codon, the pattern of response to mutations in the +4 and +5 positions was qualitatively similar, but the quantitati ve effects were smaller. Thus the optimum downstream context for initi ation is A/CUGGAU.