Transcription activation at class II CRP-dependent promoters:: identification of determinants in the C-terminal domain of the RNA polymerase α subunit
Transcription activation at class II CRP-dependent promoters:: identification of determinants in the C-terminal domain of the RNA polymerase α subunit
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DOI：
10.1093/emboj/17.12.3439
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发表时间：
1998-06-15
期刊：
影响因子：
11.4
通讯作者：
Busby, SJW
中科院分区：
文献类型：
--
作者：
Savery, NJ;Lloyd, GS;Busby, SJW
许多转录因子，包括大肠杆菌环AMP受体蛋白（CRP），通过与RNA聚合酶直接接触而起作用。在II类依赖CRP的启动子中，CRP通过两种接触激活转录：(i)与RNA聚合酶α亚基c末端结构域（α CTD）相互作用，促进RNA聚合酶与启动子DNA的初始结合；以及（ii）与RNA聚合酶α亚基n端结构域的相互作用，促进随后的启动子打开。我们使用随机诱变和丙氨酸扫描来鉴定α - CTD中II类crp依赖启动子转录激活的决定因素。我们的结果表明，II类：crp依赖转录需要残基265、271、285-288和317的侧链。在aCTD上，残基285-288和317组成了一个离散的20 x 10埃表面，在这个决定因素内的取代减少或消除了α亚基与CRP之间的合作相互作用，但不影响α亚基与DNA的结合。我们提出，在RNA聚合酶、CRP和II类CRP依赖启动子的三元复合物中，这个决定因素在α CTD中直接与CRP相互作用。并且与在I类crp依赖转录中提出的α CTD-CRP相互作用的决定因素截然不同。
Many transcription factors, including the Escherichia coli cyclic AMP receptor protein (CRP), act by making direct contacts with RNA polymerase. At Class II CRP-dependent promoters, CRP activates transcription by making two such contacts: (i) an interaction with the RNA polymerase alpha subunit C-terminal domain (alpha CTD) that facilitates initial binding of RNA polymerase to promoter DNA; and (ii) an interaction with the RNA polymerase alpha subunit N-terminal domain that facilitates subsequent promoter opening. We have used random mutagenesis and alanine scanning to identify determinants within alpha CTD for transcription activation at a Class II CRP-dependent promoter, Our results indicate that Class II: CRP-dependent transcription requires the side chains of residues 265, 271, 285-288 and 317. Residues 285-288 and 317 comprise a discrete 20 x 10 Angstrom surface on aCTD, and substitutions within this determinant reduce or eliminate cooperative interactions between alpha subunits and CRP, but do not affect DNA binding by alpha subunits, We propose that, in the ternary complex of RNA polymerase, CRP and a Class II CRP-dependent promoter, this determinant in alpha CTD interacts directly with CRP, and is distinct from and on the opposite face to the proposed determinant for alpha CTD-CRP interaction in Class I CRP-dependent transcription.