STRUCTURAL-ANALYSIS OF THE BACILLUS-SUBTILIS DELTA-FACTOR - A PROTEIN POLYANION WHICH DISPLACES RNA FROM RNA-POLYMERASE
STRUCTURAL-ANALYSIS OF THE BACILLUS-SUBTILIS DELTA-FACTOR - A PROTEIN POLYANION WHICH DISPLACES RNA FROM RNA-POLYMERASE
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DOI：
10.1006/jmbi.1995.0487
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发表时间：
1995-09-15
影响因子：
5.6
通讯作者：
HELMANN, JD
中科院分区：
文献类型：
--
作者：
DESARO, FJL;WOODY, AYM;HELMANN, JD
来自枯草芽孢杆菌的RNA聚合酶是一种复杂的混合物，包含共同的核心（beta beta'alpha（2））、20.4 kDa的delta（delta）蛋白和几种sigma（sigma）特异性因子之一。δ蛋白，连同几个截短的变体，已过量生产和纯化大肠杆菌。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，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（delta N）含有32%的α螺旋和16%的β折叠。相比之下，含有羧基末端区域（Δ C）的8.5kDa胰蛋白酶片段在很大程度上是非结构化的并且是高度带电的（净电荷为-47）。从a B纯化的RNA聚合酶。在δ基因（rpoE：：cat）中具有插入的枯草杆菌突变体含有截短的δ蛋白，表明氨基末端结构域在体内是稳定的并且含有核心结合功能。添加δ，而不是sigma（A）或δ N，置换与二元复合物中的RNA聚合酶结合的RNA。delta有效置换RNA的能力需要氨基末端核心结合结构域和聚阴离子羧基末端区域的活性。虽然Δ C也可以从RNA聚合酶中置换核酸，但这种活性需要加入大量摩尔过量的蛋白质，并且相对非特异性，因为DNA和RNA都被置换。这表明氨基端结构域的功能是结合和定向RNA聚合酶表面上的羧基端区域。(C)1995年学术出版社
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 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. (C) 1995 Academic Press Limited