An intrinsic control element for translational initiation in class 1 integrons.
Integrons are genetic elements able to capture anti-biotic resistance and other genes and to promote their transcription. Here, we have investigated integron-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.
Hanau-Berçot
Béatrice
B
INSERM EMI 0004 - LRMA, Université Paris VI, 15, rue de l'Ecole de Médecine, 75270 Paris Cedex 06, France.
Podglajen
Isabelle
I
Casin
Isabelle
I
Collatz
Ekkehard
E
D000595:Amino Acid Sequence; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics*; D019426:Integrases / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames; D010442:Peptide Chain Initiation, Translational; D010957:Plasmids
D003062:Codon; D017931:DNA Primers; D019426:Integrases