An intrinsic control element for translational initiation in class 1 integrons

Summary Integrons are genetic elements able to capture anti- biotic resistance and other genes and to promote their transcription. Here, we have investigated inte- gron-dependent translation of an aminoglycoside 6¢-N-acetyltransferase gene (aac(6¢)-Ib7) inserted at the attI1 site. N-terminal sequencing revealed that translation of this gene was initiated at a GTG codon, which is not part of a plausible translation initiation region (TIR). A short open reading frame (called ORF-11) overlapping the attI1 site was probed by site-directed mutagenesis for its contribution to aac(6¢)-Ib7 translation. When ORF-11 and its TIR were deleted en bloc, translational efficiency dropped by over 80%, as determined with an acetyltransferase- luciferase fusion product. Invalidation of the ATG start codon of ORF-11 or its putative Shine-Dalgarno sequence resulted in a decrease of over 60%, whereas the decrease was much less pronounced when the amino acid sequence of the putative ORF- 11-encoded peptide was altered or when the distance between ORF-11 and aac(6¢)-Ib7 was doubled. This demonstrates that aac(6¢)-Ib7 translation is dependent upon the translation of ORF-11, but almost certainly not upon the corresponding peptide. These results lead us to conclude that an intrinsic short ORF present in the 5¢-conserved segment of many class 1 integrons may substantially enhance expression at the translational level of captured TIR-deficient anti- biotic resistance genes.