AN RNA-POLYMERASE MUTANT WITH REDUCED ACCURACY OF CHAIN ELONGATION
AN RNA-POLYMERASE MUTANT WITH REDUCED ACCURACY OF CHAIN ELONGATION
复制标题
DOI：
10.1021/bi00368a013
复制
发表时间：
1986-10-07
期刊：
影响因子：
2.9
通讯作者：
LOEB, LA
中科院分区：
文献类型：
--
作者：
BLANK, A;GALLANT, JA;LOEB, LA
分离到一种新的大肠杆菌RNA聚合酶突变体，该突变体在体内和体外表现出链延伸的准确性降低。新的分离程序包括同时选择利福平耐药性和筛选早期的，强极性的lacZ无义突变，一个特殊的突变类之一，整个泄漏主要反映转录，而不是翻译错误的泄漏增加。由此分离的自发突变体显示这类两种不同lacZ突变的泄漏增加3-4倍。转导分析表明，定位在β-淀粉样蛋白的rpoB基因中或非常接近rpoB基因的单个突变，RNA聚合酶的亚基，赋予利福平耐药和增加无义泄漏。在体外保真度测定中，来自突变体和亲本菌株的均质RNA聚合酶显示出1/0.90 × 10 - 4的误差率。105和1/2.0 ×105，分别为poly[d（A-T）]·poly[d（A-T）]-定向的非互补GMP的错误掺入。通过产物分析验证了这些错误率，产物分析进一步揭示了在聚[r（A-U）]的合成中GMP被错误地掺入代替AMP。来自不同来源的野生型K12 RNA聚合酶的错误率为1/2.0x。105，而杂合RNA聚合酶，含有突变核心酶和野生型rho.亚基，为1/0.64 ×105.这些错误率证实了转录准确性突变体的选择。观察到的错误频率远低于其他体外试验中报告的错误频率。用于避免人为增强的错误掺入，从而定量降低错误率的保障措施进行了讨论。
A new Escherichia coli RNA polymerase mutant was isolated which exhibited reduced accuracy of chain elongation in vivo and in vitro. The novel isolation procedure consisted of simultaneous selection for rifamipicin resistance and screening for increased leakiness of an early, strongly polar nonsense mutation of lacZ, one of a special class of mutations whole leakiness reflects mainly transcriptional rather than translational errors. The spontaneous mutant thus isolated displayed a 3-4-fold increase in the leakiness of two different lacZ mutations of this class. Transduction analysis indicated that a single mutation, mapping in or very near the rpoB gene for the .beta. subunit of RNA polymerase, conferred both rifamipin resistance and increased nonsense leakiness. In an in vitro fidelity assay, homogeneous RNA polymerases from the mutant and parent strains exhibited error rates of 1/0.90 .times. 105 and 1/2.0 .times. 105, respectively, for the poly[d(A-T)].cntdot.poly[d(A-T)]-directed misincorporation of noncomplementary GMP. These error rates were verified by product analysis which further revealed that GMP was misincorporated in place of AMP in the synthesis of poly[r(A-U)]. The error rate of wild-type K12 RNA polymerase from a different source was 1/2.0 .times. 105, while that of a hybrid RNA polymerase, containing mutant core enzyme and wild-type .rho. subunit, was 1/0.64 .times. 105. These error rates confirmed the selection of a transcriptional accuracy mutant. The error frequencies observed are much lower than those reported in other in vitro assays. The safeguards used to avoid artifactually enhanced misincorporation, and to thereby quantitate lower error rates, are discussed.