Abstract
The bacterial transposon Tn7 is distinguished by its ability to recognize a specific site called attTn7, and insert just downstream of the highly conserved chromosomal glmS gene. TnsD is one of four transposon-encoded polypeptides (TnsABC+D) required for site-specific insertion of Tn7 into attTn7, and is the target site-selector that binds to a highly conserved sequence in the end of the glmS protein coding region. In this study, we identified important nucleotides within this region that are crucial for TnsD-attTn7 interaction. We also probed the regions of TnsD that interact with attTn7 and found that there are important DNA-binding determinants throughout the entire length of the protein, including an amino-terminal CCCH zinc-finger motif. A key role of TnsD is to recruit the non-sequence specific DNA-binding protein TnsC to attTn7; TnsC also interacts with and controls both the TnsA and TnsB subunits of the Tn7 transposase. TnsC stimulates the binding of TnsD to attTn7 in vivo, and TnsCD and TnsD can also interact in the absence of DNA and localize their interaction domains to the N-terminal region of each protein.
Highlights
Tn7 is a very distinctive bacterial transposon that encodes five transposition proteins: Tns A, B, C, D and E [1]
Defining important base pairs in the tnsD mutants pCW23 (TnsD) binding site Tn7 insertion occurs by the attack of the 3’OH ends of Tn7 on staggered positions on the top and bottom strands in attTn7, and DNA repair of the resulting gaps, which results in 5 bp attTn7 duplications flanking the newly inserted Tn7
The minimal E. coli 36 bp TnsD-binding site that has been defined by footprinting studies extends from attTn7 +23 to +58, and can promote maximum insertion activity in vivo and in vitro (Figure 1) [6]
Summary
Tn7 is a very distinctive bacterial transposon that encodes five transposition proteins: Tns A, B, C, D and E [1]. Whereas most transposons insert relatively randomly into many different sites, Tn7 transposition is quite specific. TnsD and TnsE are alternative target site-selectors that direct Tn7 transposition into either of two different target DNAs [2]: a very specific chromosomal attachment site or DNAs undergoing DNA replication [3]. When TnsD is the target selector, Tn7 inserts at high frequency into a specific chromosomal site called an attachment site, attTn7 [4]. Tn7 can access this highly conserved ‘safe haven’ insertion site with no obvious fitness costs to the host. Tn7 inserts into attTn7 because TnsD recognizes highly conserved sequences within the protein coding region of glmS [4], and recruits the rest of the transposition machinery to this site
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