Kimura M, Ishihama A
Department of Molecular Genetics, National Institute of Genetics, Shizuoka, Japan.
J Mol Biol. 1995 Dec 1;254(3):342-9. doi: 10.1006/jmbi.1995.0621.
The alpha subunit of Escherichia coli RNA polymerase plays a key role in assembly of the core enzyme. In previous studies the amino-terminal domain consisting of 215 amino acid residues between positions 21 and 235 was identified to be involved in this assembly, and the sites for beta and beta' association were suggested to be located within or near the two conserved regions in this amino-terminal assembly domain of alpha. For detailed functional mapping, Ala was substituted for 26 highly conserved amino acids around residues 40, 80 and 170 to 210. The alpha-point mutants were analyzed in vitro for their abilities to form dimers and to assemble beta beta' subunits. New types of assembly-deficient mutants were identified: alpha-R45A (having substituted Ala for Arg at residue 45) dimerized but did not assemble beta (and beta') subunits; and alpha-L48A showed a decreased level of alpha 2 beta subassembly formation, indicating that this region (residues 45 to 48) is responsible for beta-binding. Isolation of two mutants, alpha-K86A and alpha-V173A, both forming alpha 2 beta but not alpha 2 beta beta' complex, confirmed our previous conclusion that two separated regions participate in beta'-binding.
大肠杆菌RNA聚合酶的α亚基在核心酶的组装过程中起着关键作用。在之前的研究中，已确定位于第21至235位之间、由215个氨基酸残基组成的氨基末端结构域参与了这一组装过程，并且推测β和β'亚基的结合位点位于α亚基这一氨基末端组装结构域的两个保守区域内或其附近。为了进行详细的功能定位，用丙氨酸取代了第40、80位以及第170至210位周围的26个高度保守的氨基酸。对α亚基点突变体进行了体外分析，以研究它们形成二聚体以及组装ββ'亚基的能力。鉴定出了新型的组装缺陷型突变体：α-R45A（在第45位氨基酸处用丙氨酸取代了精氨酸）能够形成二聚体，但无法组装β（和β'）亚基；α-L48A显示α2β亚组装体的形成水平降低，这表明该区域（第45至48位氨基酸）负责β亚基的结合。分离得到的两个突变体α-K86A和α-V173A，都能形成α2β但不能形成α2ββ'复合物，这证实了我们之前的结论，即两个分开的区域参与β'亚基的结合。
