Genetic analyses of the interactions of the IS1-encoded proteins with the left end of IS1 and its insertion hotspot

Abstract

Insertion sequence IS1 specifies the InsA, ΔInsA-B′-InsB and InsA-B′-InsB protein species. These three proteins have the identical α-helix-turn-α-helix motif that is likely to be responsible for DNA binding. In fact, InsA binds to the ends of IS1, and regulates gene expression and transposition of IS1. ΔInsA-B′-InsB and/or InsA-B′-InsB has been thought to possess a transposase-like activity. Here, I examined the actions of these proteins in vivo on the promoter (pinsL) in the left end of IS1. InsA repressed pinsL-driven gene expression, both in cis and in trans. ΔInsA-B′-InsB inhibited it efficiently only when pinsL was located near the construct where ΔInsA-B′-InsB is expressed. Furthermore, it has been shown that the possible −10 sequence of pinsL is required for ΔInsA-B′-InsB to act on, but the −35 sequence where InsA binds specifically, is not. InsA-B′-InsB appeared not to work on a nearby pinsL. The cis-action of ΔInsA-B′-InsB is consistent with the previous observation that the IS1 transposase acts preferentially in cis. Interestingly, ΔInsA-B′-InsB acted on a nearby P3 promoter in the IS1 insertion hotspot, and on another promoter outside the hotspot. ΔInsA-B′-InsB may generally interact with the regions in or around promoters owing to their low DNA helix stability. Note that IS1 transposes preferentially into A + T-rich DNA segments, and that DNA is unwound from the −10 region of a promoter in transcription. The cis-preference of ΔInsA-B′-InsB would result in an overall reduction of transposition of IS1 and its defective copy in a cell, allowing stable existence of the element in its bacterial host.