Bias between the left and right inverted repeats during IS911 targeted insertion

Area of Science:

• Molecular Biology
• Genetics
• Bacterial Transposition

Background:

• IS911 is a bacterial insertion sequence with two open reading frames (ORFs) encoding transposase (OrfAB) and a regulatory protein (OrfA).
• It is flanked by left and right inverted repeats (IRL and IRR) with sequence variations.
• IS911 transposition is an asymmetric process involving the formation of an insertion sequence (IS) circle for insertion into target DNA.

Purpose of the Study:

• To investigate the functional differences between the left and right inverted repeats (IRL and IRR) of IS911.
• To determine if these differences influence the transposition process in vivo.
• To correlate transposase binding affinity with the observed functional asymmetry.

Main Methods:

• Electromobility shift assays (EMSA) were used to assess the binding affinity of a truncated transposase [OrfAB(1-149)] to IRL and IRR.
• In vivo transposition assays were performed to evaluate the efficiency of IRL and IRR in different steps of the transposition process.

Main Results:

• The truncated transposase OrfAB(1-149) demonstrated a higher binding affinity for IRR compared to IRL.
• No significant difference in inverted repeat (IR) activity was observed in the early stages of transposition.
• IRR proved more efficient than IRL in the later, insertion steps of transposition.

Conclusions:

• The two inverted repeats (IRL and IRR) of IS911 are functionally non-equivalent during transposition.
• Differential binding affinities of the transposase to IRL and IRR contribute to the observed asymmetry.
• This asymmetry likely plays a role in the ordered assembly of protein-DNA complexes during IS911 transposition.