Effect of lysine modification on the activity of the sigma subunit of Escherichia coli RNA polymerase.
Effect of lysine modification on the activity of the sigma subunit of Escherichia coli RNA polymerase.
复制标题
赖氨酸修饰对大肠杆菌RNA聚合酶sigma亚基活性的影响。
DOI：
10.1021/bi00267a012
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发表时间：
1982
期刊：
影响因子：
2.9
通讯作者：
Krakow,JS
中科院分区：
文献类型：
--
作者：
Narayanan,CS;Krakow,JS
奇坦帕利湾Narayanan和Joseph S.克拉科夫 * 摘要：用三硝基苯磺酸（TNBS）对大肠杆菌RNA聚合酶α亚基的赖氨酰残基进行化学修饰，研究了赖氨酰残基的功能。与TNBS反应后，对修饰的α的分析表明，三硝基苯化仅限于赖氨酰残基的e-氨基。在T7 DNA指导的反应中，通过刺激RNA聚合酶核心酶的能力测定，三硝基苯基化活性逐渐丧失，随后增加。五个赖氨酰基团的修饰导致活性的完全丧失。动力学分析表明，一个赖氨酰基团是关键的功能。TNP-cr能与Cr形成一种全酶复合物，其结合亲和力与Cr相当。通过使用来自T5 DNA的i/mdIII片段进行启动子识别研究。TNP-cr核心复合物不能与T5启动子形成紧密的二元复合物。通过使用d（AT）n和T7 DNA模板进行RNA链起始的研究。TNP-cr不能通过核心聚合酶刺激RNA链起始。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分析了金黄色葡萄球菌V8蛋白酶对TNP-cr或cr的限制性蛋白水解酶。结果表明，三硝基苯基化后铬的构象发生了变化。大肠杆菌DNA依赖性RNA聚合酶由三个亚基（α 2，β 3，0 ′）和第四个可解离亚基（α r）组成的催化活性核心单元组成。来自纽约城市大学亨特学院生物科学系，纽约，纽约10021。1982年5月20日收到。这项工作得到了美国国立卫生研究院（GM 18673）的研究资助。通过影响启动子识别和增加RNA链起始的速率来调节转录速率。Cr对DNA位点选择的影响可能部分归因于全酶中Cr与启动子的直接相互作用（Simpson，1979; Kudo & Doi，1981）。或者，cr的结合可以诱导全酶中的特异性构象，导致对启动子位点的亲和力增加和对DNA一般位点的亲和力降低（Wu等人，1976年）。RNA链起始于双链DNA中的切口处，见核心
Chittampalli S. Narayanan and Joseph S. Krakow* abstract: The function of lysyl residues of the a subunit of the RNA polymerase from Escherichia coli was investigated by chemicalmodification with trinitrobenzenesulfonic acid (TNBS). Followingreaction with TNBS, analysis of the modified a indicated that trinitrophenylation was limited to the e-amino groups of lysyl residues. Progressive loss in the activity of a followed increasing trinitrophenylation as assayed by the ability to stimulate RNA polymerase core enzyme in a reaction directed by T7 DNA. Modification of five lysyl groups resulted in the complete loss of a activity. Kinetic analysis indicated that one lysyl group is critical for the function of a. TNP-cr was able to form a holoenzyme complex with a binding affinity comparable to thatof cr. Promoter recognition studies were done by using i/mdlll fragments from T5 DNA. The TNP-cr core complex was unable to form a tight binary complex with the T5 promoters. Studies on RNA chain initiation were carried out by using d (AT)„and T7 DNA templates. TNP-cr was unable to stimulate RNA chain initiation by core polymerase. Limited proteolytic digests of TNP-cr or cr using Staphylococcus aureus V8 protease were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results suggested a change in the con-formation of cr following trinitrophenylation. e Escherichia coli DNA-dependent RNA polymerase consists of a catalytically competent core unit composed of three subunits (a2,/3, 0') and a fourth dissociable subunit (cr). The presence of cr in the holoenzyme stimulates the overall f From the Department of Biological Sciences, Hunter College of the City University of New York, New York, New York 10021. Received May 20, 1982. This work was supported by a research grantfrom the National Institutes of Health (GM 18673). rate of transcription by effecting promoter recognition and increasing the rate of RNA chain initiation. The effect of cr on DNA site selection could be due, in part, to a direct interaction of cr in the holoenzyme with the promoter (Simpson, 1979; Kudo & Doi, 1981). Alternatively, binding of cr may induce a specific conformation in the holoenzyme resulting in an increased affinity for promoter sites and a lowered af-finity for DNA general sites (Wu et al., 1976). RNA chain initiation at nicks in double-stranded DNA seen with the core