Weinreich M D, Reznikoff W S
Department of Biochemistry, University of Wisconsin-Madison 53706.
J Bacteriol. 1992 Jul;174(14):4530-7. doi: 10.1128/jb.174.14.4530-4537.1992.
The Fis (factor for inversion stimulation) protein of Escherichia coli was found to influence the frequency of transposon Tn5 and insertion sequence IS50 transposition. Fis stimulated both Tn5 and IS50 transposition events and also inhibited IS50 transposition in Dam-bacteria. This influence was not due to regulation by Fis of the expression of the Tn5 transposition proteins. We localized, by DNase I footprinting, one Fis site overlapping the inside end of IS50 and give evidence to strongly suggest that when Fis binds to this site, IS50 transposition is inhibited. The Fis site at the inside end overlaps three Dam GATC sites, and Fis bound efficiently only to the unmethylated substrate. Using a mobility shift assay, we also identified another potential Fis site within IS50. Given the growth phase-dependent expression of Fis and its differential effect on Tn5 versus IS50 transposition in Dam-bacteria, we propose that the high levels of Fis present during exponential growth stimulate transposition events and might bias those events toward Tn5 and away from IS50 transposition.
已发现大肠杆菌的Fis(反转刺激因子)蛋白会影响转座子Tn5和插入序列IS50的转座频率。Fis既刺激Tn5和IS50的转座事件,也在Dam⁻细菌中抑制IS50转座。这种影响并非源于Fis对Tn5转座蛋白表达的调控。我们通过DNA酶I足迹法定位到一个与IS50内侧末端重叠的Fis位点,并提供有力证据表明,当Fis结合到该位点时,IS50转座受到抑制。内侧末端的Fis位点与三个Dam GATC位点重叠,且Fis仅能有效结合未甲基化的底物。利用迁移率变动分析,我们还在IS50内鉴定出另一个潜在的Fis位点。鉴于Fis的表达依赖生长阶段,且其对Dam⁻细菌中Tn5与IS50转座有不同影响,我们提出指数生长期间存在的高水平Fis会刺激转座事件,并可能使这些事件偏向Tn5,远离IS50转座。