Chemical modifications of the sigma subunit of the E. coli RNA polymerase.
Chemical modifications of the sigma subunit of the E. coli RNA polymerase.
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大肠杆菌 RNA 聚合酶 sigma 亚基的化学修饰。
DOI：
10.1093/nar/11.9.2701
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发表时间：
1983
影响因子：
14.9
通讯作者：
Krakow,JS
中科院分区：
文献类型：
--
作者：
Narayanan,CS;Krakow,JS
采用基团特异性试剂对sigma的精氨酸、半胱氨酸和羧基氨基酸（谷氨酸和天冬氨酸）残基进行了化学修饰。用苯乙二醛修饰3个精氨酸残基或用n -乙基马来酰亚胺（NEM）修饰3个半胱氨酸残基后，sigma活性丧失。失活的动力学数据分析表明，一个精氨酸或半胱氨酸残基是sigma活性所必需的。在低NEM浓度下，烷基化仅限于非临界半胱氨酸，鉴定为半胱氨酸-132。精氨酸或半胱氨酸残基的修饰对失活的sigma与核心聚合酶的结合没有明显的影响。用水溶性碳二酰亚胺(l-乙基-3-（3-二甲氨基丙基）碳二酰亚胺盐酸盐（EDC）或l-环己基-3-(2-morpholinoethyl)碳二酰亚胺甲对甲苯磺酸盐（CMC）修饰天冬氨酸和/或谷氨酸残基，导致sigma活性丧失。失活数据表明，一个羧基氨基酸残基是sigma活性所必需的。在甘氨酸存在的情况下，EDC、CMC或EDC修饰的Sigma对支持启动子结合和核心聚合酶的起始不起作用。与EDC + (3H)甘氨酸反应导致甘氨酸并入sigma。（3H）甘氨酸-西格玛不能形成稳定的全酶复合物。
Ihe Junction or arginlne, cystelne and carboxylic amino acid (glutamic and aspartic) residues of sigma was studied using chemical modification by group specific reagents. Following modification of 3 arginine residues with phenylglyoxal or 3 cysteine residues with N-ethylmaleimide (NEM) sigma activity was lost. Analysis of the kinetic data for inactivation indicated that one arginlne or cysteine residue is essential for sigma activity. At low NEM concentration alkylation was limited to a non-critical cysteine which was identified as cysteine-132. Modification of arginine or cysteine residues had no observable effect on the binding of the inactivated sigma to the core polymerase. Modification of aspartic and/or glutamic acid residues with the water-soluble carbodiimides l-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride (EDC) or l-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMC) resulted in loss of sigma activity. The inactivation data indicated that one carboxylic amino acid residue is essential for sigma activity. Sigma modified with EDC, CMC or EDC in the presence of glycine was inactive in supporting promoter binding and initiation by core polymerase. Reaction with EDC plus (3H)glycine resulted in the incorporation of glycine into sigma. The (3H)glycine-sigma was unable to form a stable holoenzyme complex.