Kainz M, Gourse R L
Department of Bacteriology, University of Wisconsin, 1550 Linden Drive, Madison, WI, 53706, USA.
J Mol Biol. 1998 Dec 18;284(5):1379-90. doi: 10.1006/jmbi.1998.2272.
We screened a collection of single alanine residue substitution mutants spanning the entire C-terminal domain of the alpha subunit (alphaCTD) of Escherichia coli RNA polymerase (RNAP) for defects in rho-dependent transcription termination at lambdatR1 in vivo and in vitro, and thereby identified a patch of amino acid residues in the alphaCTD required for efficient rho-dependent termination. NusA addition led to the stimulation of rho-dependent termination under our conditions in vitro. The termination defects of a few mutant RNAPs could be attributed to altered interactions with the NusA protein, but rho-dependent termination by most of the defective RNAPs was still stimulated normally by NusA. The NusA-enhanced transcription pausing behaviors of the mutant RNAPs did not always correlate with their rho-dependent termination phenotypes. We conclude that the alphaCTD is a target for interactions with NusA that influence both termination and pausing, but in addition it participates in rho-dependent transcription termination in a NusA-independent manner.
我们筛选了一系列单丙氨酸残基取代突变体，这些突变体覆盖了大肠杆菌RNA聚合酶（RNAP）α亚基的整个C末端结构域（αCTD），以检测其在体内和体外λtR1处ρ依赖性转录终止的缺陷，从而在αCTD中鉴定出了有效ρ依赖性终止所需的一片氨基酸残基。在我们的体外条件下，添加NusA会导致ρ依赖性终止的刺激。一些突变体RNAP的终止缺陷可归因于与NusA蛋白相互作用的改变，但大多数有缺陷的RNAP的ρ依赖性终止仍能被NusA正常刺激。突变体RNAP的NusA增强转录暂停行为并不总是与其ρ依赖性终止表型相关。我们得出结论，αCTD是与NusA相互作用的靶点，这种相互作用影响终止和暂停，但此外，它还以不依赖NusA的方式参与ρ依赖性转录终止。
