The structure and functions of protein RepA of plasmid Rtsl

RepA蛋白编码在mini-Rtsl基因组上，是质粒Rtsl复制所必需的，由288个氨基酸残基组成。RepA的纯化和蛋白质与DNA的结合。从含有重组质粒的大肠杆菌细胞裂解液中制备纯化的RepA。利用DNA印迹法研究纯化的RepA与mini-Rtsl DNA的结合，发现RepA特异性结合inci和inci内含子以及RepA启动子的上游2。repa突变体的构建及其功能的改变。1) repA中间的突变。我们利用位于repA基因中间独特的StyI和XbaI位点，将一个4氨基酸插入到repA蛋白的框架中。两种RepA突变蛋白都失去了启动蛋白功能，但保留了很强的自抑制蛋白不相容功能。2) repA 3'端突变。通过位点定向诱变获得了7个RepA突变体，每个突变体在RepA的c端区域含有单个氨基酸替代或小缺失。(a) Lys268对启动物和自抑制物不相容功能都很重要。(b) Arg279只参与启动器功能。(c)从c端删除四个氨基酸残基导致质粒的高拷贝数。(d)从c端删除5个氨基酸残基恢复了野生型RepA表型。

RepA protein, encoded on the mini-Rtsl geneome and essential for the plasmid Rtsl replication, is composed of 288 amino acid residues.1. Purification of RepA and binding of the protein to DNA.Purified RepA was prepared from the E. coli cell-lysate harboring the recombinant plasmid with repA. Binding of the purified RepA to mini-Rtsl DNA was studied by DNaseI foot printing method, and revealed that RepA binds specifically to inci and incII iterons as well as to the immediately upstream of the repa promoter.2. Construction of repa mutants and their altered functions.1) Mutations in the middle of repA. We utilized the unique StyI and XbaI sites that located in the middle of repA gene to insert a 4-amino-acid in frame to RepA protein. Both RepA mutant proteins lost initiator function but retained strong autorepressor-incompatibility functions.2) Mutations in the 3' end of repA. Seven RepA mutants, each of which contained a single amino acid substitution or small deletion in the C-terminal region of RepA, were obtained by site-directed mutagenesis, The following findings were obtained.(a) Lys268 is important for both initiator and autorepressor-incompatibility functions. (b) Arg279 is involved in only initiator function. (c) Deletion of four amino acid residues from the C-terminus induced a high copy number of the plasmid. (d) Deletion of five amino acid residues from the C-terminus restored the wild type RepA phenotypes.