Department of Biochemistry, University of Western Ontario, London, Ontario N6A 5C1, Canada.
Mob DNA. 2012 Apr 13;3:7. doi: 10.1186/1759-8753-3-7.
The H-NS protein is a global regulator of gene expression in bacteria and can also bind transposition complexes (transpososomes). In Tn5 transposition H-NS promotes transpososome assembly in vitro and disruption of the hns gene causes a modest decrease in Tn5 transposition (three- to five-fold). This is consistent with H-NS acting as a positive regulator of Tn5 transposition. Molecular determinants for H-NS binding to the Tn5 transpososome have not been determined, nor has the strength of the interaction been established. There is also uncertainty as to whether H-NS regulates Tn5 transposition in vivo through an interaction with the transposition machinery as disruption of the hns gene has pleiotropic effects on Escherichia coli, the organism used in this study.
In the current work we have further examined determinants for H-NS binding to the Tn5 transpososome through both mutational studies on Tn5 termini (or 'transposon ends') and protein-protein cross-linking analysis. We identify mutations in two different segments of the transposon ends that abrogate H-NS binding and characterize the affinity of H-NS for wild type transposon ends in the context of the transpososome. We also show that H-NS forms cross-links with the Tn5 transposase protein specifically in the transpososome, an observation consistent with the two proteins occupying overlapping binding sites in the transposon ends. Finally, we make use of the end mutations to test the idea that H-NS exerts its impact on Tn5 transposition in vivo by binding directly to the transpososome. Consistent with this possibility, we show that two different end mutations reduce the sensitivity of the Tn5 system to H-NS regulation.
H-NS typically regulates cellular functions through its potent transcriptional repressor function. Work presented here provides support for an alternative mechanism of H-NS-based regulation, and adds to our understanding of how bacterial transposition can be regulated.
H-NS 蛋白是细菌中基因表达的全局调控因子，也可以结合转座复合物（转座体）。在 Tn5 转座中，H-NS 促进转座体在体外组装，而 hns 基因的破坏导致 Tn5 转座的适度减少（三到五倍）。这与 H-NS 作为 Tn5 转座的正调控因子的作用一致。尚未确定 H-NS 结合 Tn5 转座体的分子决定因素，也未确定相互作用的强度。也不确定 H-NS 是否通过与转座机制的相互作用来调节体内 Tn5 转座，因为 hns 基因的破坏对本研究中使用的大肠杆菌等生物体有多种影响。
在目前的工作中，我们通过对 Tn5 末端（或“转座子末端”）的突变研究和蛋白质-蛋白质交联分析，进一步研究了 H-NS 结合 Tn5 转座体的决定因素。我们确定了转座子末端的两个不同区域的突变，这些突变消除了 H-NS 的结合，并在转座体的背景下表征了 H-NS 对野生型转座子末端的亲和力。我们还表明，H-NS 与 Tn5 转座酶蛋白特异性地在转座体中形成交联，这一观察结果与这两种蛋白质在转座子末端占据重叠的结合位点一致。最后，我们利用末端突变来测试 H-NS 通过直接结合转座体来发挥其对体内 Tn5 转座的影响的想法。与这种可能性一致，我们表明两种不同的末端突变降低了 Tn5 系统对 H-NS 调节的敏感性。
H-NS 通常通过其强大的转录抑制剂功能来调节细胞功能。这里提出的工作为 H-NS 为基础的调节的另一种机制提供了支持，并增加了我们对细菌转座如何被调节的理解。
