作　　者： ; ;

机构地区： 中山医科大学

出　　处： 《生物化学杂志》 1990年第4期314-320,共7页

摘　　要： E.coli RNA聚合酶ββ'亚单位编码的基因rpoBC与核糖核蛋白基因rplJL共同构成rpoBC操纵子。rpl-rpo间区转录终止信号~tL7的转录产物RNA有两组富含C-G的反向对称结构及一串寡聚U;反向对称区可形成1:2和3:4茎环或单一5:6茎环。 用M13mp11噬菌体插入~tL7的112bp片段重组M13mp11-490,在此基础上用定点突变技术建立~tL7的七核苷酸缺失突变体,从而破坏1:2茎环,建成M13mp11-85。 分别把原型~tL7及缺失型~tL7~v克隆到表达质粒pHR24中,构建成pHR37(P_ftsQ^tL7-galk)及pHR24-9(p_ftsQ-~tL7~v-galk)。测定galk基因产物半乳糖激酶活性,推算转录的终止效率。结果表明:~tL7终止效率为50％,~tL7~v为20％。说明仅有3:4茎环及寡聚U,即具终止作用; 1:2茎环通过某种方式加强3:4茎环从而提高终止效率。 rpoBC operon of E.coli includes rpoBC gene, coding for ββ' subunits of RNA polymerase and its up-stream gene rplJL coding for ribosomal proteins. The intergenic region between rpl-rpo lies tL7, a transcriptional terminator. Transcripts of tL7 show two dyad symmetry G-C-rich areas followed by a poly-U sequence, these G-C rich areas form either 1:2 plus 3:4 stem-loops, or a single 5:6 stem-loops.The 112bp fragment of tL7 has been inserted into Ml3mp11 vector to form M13mpll-490.A seven nucleotide-deletion mutant, M13mpll-85,based on M13mpll-490 as template, was constructed using site-directed mutagenesis technique. In M13-mpll-85,the first arm of 1:2 stem-loop has been destroyed because of the deletion.The wild type tL7 and the deleted tL7 (tL7v) was cloned into pHR24, an expressive plasmid derived from pKO-1 system. Thus pHR37(PftsQ-tL7-galK)and pHR24-9(PftsQ-tL7v-galK) were constructed.Using 14C-galactose as substrate, the activities of galactokinase, which is the gene expression poduct of galk, can be meassured.The transcriptional termination efficiencies of tL7 and tL7v were calculated separately via transcriptional levels of galk.Our studies showed a 50% transcriptional termination efficiency in tL7, while only 20% in tL7v. This result suggested that, an effective termination was contributed by 3:4 stem-loop together with its down-stream polyU. With the existence of 1:2 stem-loop, 3:4 stem-loop may be somehow strengthened and a much higher terminational efficiency was obtained.

关 键 词： 定点突变 转录终止 操纵子

领　　域： [生物学]