López de Saro F J, Woody A Y, Helmann J D
Section of Microbiology, Cornell University, Ithaca, NY 14853-8101, USA.
J Mol Biol. 1995 Sep 15;252(2):189-202. doi: 10.1006/jmbi.1995.0487.
RNA polymerase from Bacillus subtilis is a complex mixture comprising a common core (beta beta' alpha 2), the 20.4 kDa delta (delta) protein, and of one of several sigma (sigma) specificity factors. The delta protein, together with several truncated variants, has been overproduced and purified from Escherichia coli. It is highly acidic (pI = 3.6) and contains two distinct regions, a 13 kDa amino-terminal domain with fairly uniform charge distribution and a glutamate and aspartate residue-rich carboxyl-terminal region. The purified amino-terminal domain (delta N) contains 32% alpha-helix and 16% beta-sheet, as judged by circular dichroism analysis. In contrast, an 8.5 kDa tryptic fragment containing the carboxyl-terminal region (delta C) is largely unstructured and highly charged (net charge of -47). RNA polymerase purified from a B. subtilis mutant with an insertion in the delta gene (rpoE::cat) contains a truncated delta protein, indicating that the amino-terminal domain is stable in vivo and contains a core-binding function. Addition of delta, but not sigma A or delta N, displaces RNA bound to RNA polymerase in a binary complex. The ability of delta to displace RNA efficiently requires the activities of both the amino-terminal core-binding domain and the polyanionic carboxyl-terminal region. Although delta C can also displace nucleic acids from RNA polymerase, this activity requires the addition of a large molar excess of protein and is relatively non specific in that both DNA and RNA are displaced. This suggests that the function of the amino-terminal domain is to bind and orient the carboxyl-terminal region on the surface of RNA polymerase.
来自枯草芽孢杆菌的RNA聚合酶是一种复杂的混合物，包含一个共同的核心（ββ'α2）、20.4 kDa的δ（delta）蛋白以及几种σ（sigma）特异性因子之一。δ蛋白与几种截短变体一起已在大肠杆菌中过量表达并纯化。它是高度酸性的（pI = 3.6），包含两个不同的区域，一个13 kDa的氨基末端结构域，电荷分布相当均匀，以及一个富含谷氨酸和天冬氨酸残基的羧基末端区域。通过圆二色性分析判断，纯化的氨基末端结构域（δN）含有32%的α-螺旋和16%的β-折叠。相比之下，一个包含羧基末端区域的8.5 kDa胰蛋白酶片段（δC）在很大程度上是无结构的且带电量很高（净电荷为 -47）。从δ基因插入突变体（rpoE::cat）的枯草芽孢杆菌中纯化的RNA聚合酶含有截短的δ蛋白，这表明氨基末端结构域在体内是稳定的且具有核心结合功能。添加δ蛋白，但不添加σA或δN，可以取代二元复合物中与RNA聚合酶结合的RNA。δ蛋白有效取代RNA的能力需要氨基末端核心结合结构域和多阴离子羧基末端区域的活性。虽然δC也可以从RNA聚合酶中取代核酸，但这种活性需要添加大量摩尔过量的蛋白质，并且相对非特异性，因为DNA和RNA都会被取代。这表明氨基末端结构域的功能是在RNA聚合酶表面结合并定向羧基末端区域。
