In vivo translational start site selection on leaderless mRNA transcribed from the Streptomyces fradiae aph gene

Abstract

The message of the Streptomyces fradiae aph gene lacks a leader sequence and therefore is translated in the absence of a conventional Shine-Dalgarno interaction between mRNA and 30S ribosomal subunits. Insertion mutations generating short leaders of 2 or 4 nucleotides on the 5' end of the aph transcript reduced translational efficiency. A 4-base leader (5'-AUGC-3') placing a potential out-of-frame start codon immediately upstream of the aph coding sequence prevented detectable translation in the aph reading frame. The upstream AUG in this mutant was able to drive the expression of a reporter gene in a translational fusion vector, implying that this start codon was utilized in favor of the downstream AUG. Additional leaders (5'-AUAUGC-3' or 5'-CAUAUGC-3') placing 2 or 3 nucleotides 5' to the upstream AUG relieved this apparent discrimination, permitting translation of the APH protein from the downstream AUG. These results suggest that the position of a start codon with respect to the 5' terminus of aph mRNA is a determinant of translational efficiency and start site selection.