An intrinsic control element for translational initiation in class 1 integrons

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Integrons are mobile genetic elements that capture and express genes, particularly antibiotic resistance genes.
• The expression of captured genes within integrons is complex and not fully understood.

Purpose of the Study:

• To investigate the mechanism of integron-dependent translation initiation for the aminoglycoside 6 -N-acetyltransferase gene (aac(6 )-Ib7).
• To determine the role of a short open reading frame (ORF-11) in the translational efficiency of aac(6 )-Ib7.

Main Methods:

• Site-directed mutagenesis was used to alter ORF-11 and its associated translation initiation region (TIR).
• Acetyltransferase-luciferase fusion assays were employed to quantify translational efficiency.
• N-terminal sequencing was performed to identify the translation start site of aac(6 )-Ib7.

Main Results:

• Translation of aac(6 )-Ib7 was initiated at a GTG codon lacking a canonical TIR.
• Deletion of ORF-11 and its TIR reduced translational efficiency by over 80%.
• Disruption of ORF-11's start codon or Shine-Dalgarno sequence decreased expression by over 60%, while altering the peptide sequence or increasing distance had minimal impact.

Conclusions:

• Translation of captured antibiotic resistance genes in integrons can be dependent on upstream short open reading frames.
• ORF-11 significantly enhances the translational efficiency of TIR-deficient genes like aac(6 )-Ib7.
• This mechanism provides a novel way for integrons to express captured antibiotic resistance genes.