Kulish D, Lee J, Lomakin I, Nowicka B, Das A, Darst S, Normet K, Borukhov S
Department of Microbiology and Immunology, State University of New York, Health Science Center at Brooklyn, New York 11203, USA.
J Biol Chem. 2000 Apr 28;275(17):12789-98. doi: 10.1074/jbc.275.17.12789.
The transcript cleavage factors GreA and GreB of Escherichia coli are involved in the regulation of transcription elongation. The surface charge distribution analysis of their three-dimensional structures revealed that the N-terminal domains of GreA and GreB contain a small and large basic "patch," respectively. To elucidate the functional role of basic patch, mutant Gre proteins were engineered in which the size and charge distribution of basic patch were modified and characterized biochemically. We found that Gre mutants lacking basic patch or carrying basic patch of decreased size bind to RNA polymerase and induce transcript cleavage reaction in minimally backtracked ternary elongation complex (TEC) with the same efficiency as the wild type factors. However, they exhibit substantially lower readthrough and cleavage activities toward extensively backtracked and arrested TECs and display decreased efficiency of photocross-linking to the RNA 3'-terminus. Unlike wild type factors, basic patch-less Gre mutants are unable to complement the thermosensitive phenotype of GreA(-):GreB(-) E. coli strain. The large basic patch is required but not sufficient for the induction of GreB-type cleavage reaction and for the cleavage of arrested TECs. Our results demonstrate that the basic patch residues are not directly involved in the induction of transcript cleavage reaction and suggest that the primary role of basic patch is to anchor the nascent RNA in TEC. These interactions are essential for the readthrough and antiarrest activities of Gre factors and, apparently, for their in vivo functions.
大肠杆菌的转录切割因子GreA和GreB参与转录延伸的调控。对其三维结构的表面电荷分布分析表明，GreA和GreB的N端结构域分别包含一个小的和大的碱性“区域”。为了阐明碱性区域的功能作用，构建了突变型Gre蛋白，其中碱性区域的大小和电荷分布被改变，并进行了生化特性分析。我们发现，缺乏碱性区域或携带减小尺寸碱性区域的Gre突变体与RNA聚合酶结合，并在最小程度回溯的三元延伸复合物（TEC）中诱导转录切割反应，其效率与野生型因子相同。然而，它们对广泛回溯和停滞的TEC表现出显著较低的通读和切割活性，并且与RNA 3'末端的光交联效率降低。与野生型因子不同，无碱性区域的Gre突变体无法互补GreA(-):GreB(-)大肠杆菌菌株的温度敏感表型。大的碱性区域对于诱导GreB型切割反应和切割停滞的TEC是必需的，但不是充分的。我们的结果表明，碱性区域残基不直接参与转录切割反应的诱导，并表明碱性区域的主要作用是将新生RNA锚定在TEC中。这些相互作用对于Gre因子的通读和抗停滞活性至关重要，显然，对于它们在体内的功能也是如此。
