The structure and functions of protein RepA of plasmid Rtsl

质粒Rtsl蛋白RepA的结构与功能

• 批准号: 63480153
• 负责人: TERAWAKI Yoshiro
• 金额: $ 2.56万
• 依托单位: Shinshu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-63480153/
• 关键词: Plasmid Rtsl; RepA protein; DNA binding; RepA mutant proteins; Replication; Autorepressor function; incompatibility; RepA蛋白; C末端領域; 部位特異変異体; 複製; 自己制御; DNA複製; 複製必須蛋白質; 変異蛋白質; 複製開始能; オートリプレッサー活性

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.

RepA蛋白编码在Mini-RTSL基因组上，对质粒RTSL复制必不可少，由288个氨基酸残基组成。1。从含有重组质粒的大肠杆菌细胞溶解液中制备了RepA的纯化和蛋白质与DNA的结合。通过DNAsei脚印方法研究了纯化的RepA与Mini-RTSL DNA的结合，并揭示了RepA与INCI和INCII ITERON以及REPA启动子的立即上游的结合。2。 REPA突变体的构建及其功能改变。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-氨基酸的框架插入repa蛋白。两种RepA突变蛋白都失去了引发剂功能，但保留了强大的自动压缩函数。2）在REPA的3'末端的突变。通过位置定向诱变获得了七个REPA突变体，每个REPA突变体在REPA的C末端区域中包含单个氨基酸取代或小缺失，获得以下发现。（a）Lys268对引发剂和自动锻炼者都很重要。 - 兼容功能。 （b）ARG279仅参与启动器功能。 （c）从C末端删除四个氨基酸残基会诱导质粒的高拷贝数。 （d）从C末端删除了五个氨基酸残基，恢复了野生型REPA表型。

项目成果

神尾好是 他: "Purification of Rts1 RepA protein and binding of the protein to mini-Rts1 DNA" Journal of Bacteriology. 170. 4411-4414 (1988)

Yoshize Kamio 等人：“Rts1 RepA 蛋白的纯化以及该蛋白与 mini-Rts1 DNA 的结合”《细菌学杂志》170. 4411-4414 (1988)。

Y.Terawaki;Z.Hong;Y.Itoh;Y.Kamio: J.Bacteriol.170. 1261-1267 (1988)

Y.Terawaki；Z.Hong；Y.Itoh；Y.Kamio：J.Bacteriol.170。

Terawaki, Y.: "Effects of mutations in the repA gene of plasmid Rtsl on plasmid replication and autorepressor function." J. Bacteriol. 172:786-792, 1990.

Terawaki, Y.：“质粒 Rtsl 的 repA 基因突变对质粒复制和自阻抑功能的影响。”

伊藤義文 他: "Replication properties of mini-Rts1 derivatives deleted for DnaA boxes in the replication origin" Plasmid. 21. 242-246 (1989)

Yoshifumi Ito 等人：“复制起点中 DnaA 盒删除的 mini-Rts1 衍生物的复制特性” 质粒 21. 242-246 (1989)

Zeng, H.: "Site-directed mutations in the repA C-terminal region of plasmid Rtsl: Pleiotropic effects on the replication and autorepressor functions." J. Bacteriol. 172 (No.5) 1990.

Zeng, H.：“质粒 Rtsl 的 repA C 端区域的定点突变：对复制和自抑制功能的多效性影响。”