The structure and functions of protein RepA of plasmid Rtsl

REPA蛋白由288个氨基酸残基组成，编码在微小的Rts1基因组上，是Rts1复制所必需的。REPA的纯化及其与DNA的结合从含有REPA的重组质粒的大肠杆菌细胞裂解液中制备纯化的REPA。用DNaseI足印方法研究了纯化的REPA与mini-Rts1 DNA的结合，结果表明，REPA与INCI和INC II启动子以及REPA启动子的上游都有特异的结合。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基因中间的独特的StyI和XbaI位点将4个氨基酸插入到REPA蛋白的框架中。两个REPA突变蛋白都失去了启动子功能，但保留了很强的自抑制不相容功能。2)REPA 3‘端突变。通过定点突变获得了7个REPA突变体，每个突变体在REPA的C-末端区域都含有一个氨基酸替换或小的缺失，得到了以下发现：(A)Lys268对启动子和自抑制子不亲和功能都是重要的。(B)Arg279只参与引发剂功能。(C)C末端4个氨基酸残基的缺失导致了高拷贝数的质粒。(D)C末端5个氨基酸残基的缺失恢复了野生型REPA的表型。

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.