Rousseau P, Loot C, Turlan C, Nolivos S, Chandler M
Laboratoire de Microbiologie et Génétique Moléculaires, CNRS-UMR5100, Université de Toulouse III, Toulouse 31062, France.
J Bacteriol. 2008 Sep;190(18):6111-8. doi: 10.1128/JB.00452-08. Epub 2008 Jun 27.
IS911 is a bacterial insertion sequence composed of two consecutive overlapping open reading frames (ORFs [orfA and orfB]) encoding the transposase (OrfAB) as well as a regulatory protein (OrfA). These ORFs are bordered by terminal left and right inverted repeats (IRL and IRR, respectively) with several differences in nucleotide sequence. IS911 transposition is asymmetric: each end is cleaved on one strand to generate a free 3'-OH, which is then used as the nucleophile in attacking the opposite insertion sequence (IS) end to generate a free IS circle. This will be inserted into a new target site. We show here that the ends exhibit functional differences which, in vivo, may favor the use of one compared to the other during transposition. Electromobility shift assays showed that a truncated form of the transposase [OrfAB(1-149)] exhibits higher affinity for IRR than for IRL. While there was no detectable difference in IR activities during the early steps of transposition, IRR was more efficient during the final insertion steps. We show here that the differential activities between the two IRs correlate with the different affinities of OrfAB(1-149) for the IRs during assembly of the nucleoprotein complexes leading to transposition. We conclude that the two inverted repeats are not equivalent during IS911 transposition and that this asymmetry may intervene to determine the ordered assembly of the different protein-DNA complexes involved in the reaction.
IS911是一种细菌插入序列，由两个连续重叠的开放阅读框（ORF，即orfA和orfB）组成，分别编码转座酶（OrfAB）和一种调节蛋白（OrfA）。这些ORF由末端左右反向重复序列（分别为IRL和IRR）界定，核苷酸序列存在若干差异。IS911转座是不对称的：每条末端在一条链上被切割以产生一个游离的3'-OH，然后该3'-OH作为亲核试剂攻击相对的插入序列（IS）末端以产生一个游离的IS环。这个环将被插入到一个新的靶位点。我们在此表明，这些末端表现出功能差异，在体内，转座过程中可能更倾向于使用其中一个末端而非另一个。电泳迁移率变动分析表明，转座酶的截短形式[OrfAB(1 - 149)]对IRR的亲和力高于对IRL的亲和力。虽然在转座早期步骤中IR活性没有可检测到的差异，但在最终插入步骤中IRR更有效。我们在此表明，两个IR之间的差异活性与转座过程中核蛋白复合物组装期间OrfAB(1 - 149)对IR的不同亲和力相关。我们得出结论，在IS911转座过程中两个反向重复序列并不等同，并且这种不对称性可能会干预以确定反应中涉及的不同蛋白质 - DNA复合物的有序组装。
