Arciszewska L K, McKown R L, Craig N L
Department of Microbiology and Immunology, University of California, San Francisco 94143.
J Biol Chem. 1991 Nov 15;266(32):21736-44.
We have purified TnsB, a transposition protein encoded by the bacterial transposon Tn7. The purification procedure involves three chromatographic steps (DNA-cellulose, norleucine-Sepharose, and phosphocellulose) and yields milligram quantities of highly purified protein. The apparent molecular mass of denatured TnsB protein is approximately 85 kDa. Gel filtration chromatography and sucrose gradient sedimentation studies indicate that in solution, native TnsB is a monomer of nonspherical shape. Using DNase I protection analysis, we established that TnsB is a sequence-specific DNA-binding protein that recognizes multiple sites in both ends of the transposon. The TnsB binding sites, three in the left end of Tn7 and four in the right end, are highly related in nucleotide sequence and are located in DNA segments that we have previously shown contain cis-acting sequences important for Tn7 transposition. Our results also show that one of the TnsB binding sites overlaps a proposed promoter for the transposition genes of Tn7. These studies suggest that the specific binding of TnsB to the ends of Tn7 mediates recombination and may also regulate the expression of Tn7-encoded transposition genes.
我们已经纯化了TnsB，一种由细菌转座子Tn7编码的转座蛋白。纯化过程包括三个色谱步骤（DNA纤维素、正亮氨酸-琼脂糖和磷酸纤维素），并能产生毫克量的高度纯化蛋白。变性的TnsB蛋白的表观分子量约为85 kDa。凝胶过滤色谱和蔗糖梯度沉降研究表明，在溶液中，天然TnsB是一种非球形单体。通过DNase I保护分析，我们确定TnsB是一种序列特异性DNA结合蛋白，可识别转座子两端的多个位点。TnsB结合位点，在Tn7左端有三个，右端有四个，在核苷酸序列上高度相关，且位于我们之前所示的含有对Tn7转座重要的顺式作用序列的DNA片段中。我们的结果还表明，TnsB结合位点之一与Tn7转座基因的一个假定启动子重叠。这些研究表明，TnsB与Tn7末端的特异性结合介导了重组，并且可能还调节了Tn7编码的转座基因的表达。
