The immediate downstream codon strongly influences the efficiency of utilization of eukaryotic translation initiation codons.

Nucleotide substitutions were introduced into the initiation site of an influenza virus NS cDNA derivative at the +4, +5 and +6 positions (where the A of the AUG codon is defined as +1), in the background of either AUG or CUG as the initiation codon. Capped transcripts of these constructs were translated in rabbit reticulocyte lysate under conditions where the selection of initiation sites conformed to the scanning ribosome model. With CUG as the initiation codon, the efficiency of initiation 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 smaller. The residues favourable to initiation were as follows: at +4, only G was stimulatory; at +5, A was strongly stimulatory and C fairly beneficial; and at +6, only U exerted any positive influence. The positive influence of the favourable residues (or the negative influence of unfavourable residues) at each position appeared to be additive. With AUG as the initiation codon, the pattern of response to mutations in the +4 and +5 positions was qualitatively similar, but the quantitative effects were smaller. Thus the optimum downstream context for initiation is A/CUGGAU.