Hanna M M, Liu K
Department of Chemistry and Biochemistry, The University of Oklahoma, Norman 73019, USA.
J Mol Biol. 1998 Sep 18;282(2):227-39. doi: 10.1006/jmbi.1998.2005.
Proteins in a partially fractionated Escherichia coli extract that interact with the nascent RNA in active transcription complexes from several promoters were detected using the photocrosslinking ribonucleotide analogs 5-(azidophenacyl)thio-UTP or 5-(azidophenacyl)thio-CTP as transcription substrates. Upon irradiation of ternary transcription complexes, several extract proteins were crosslinked to the RNA. Most notably, a small protein was crosslinked to the RNA in complexes on seven of nine templates tested. This protein was purified and sequenced and found to match a hypothetical protein, MsmC/CspE, recently shown to be involved in chromatin partitioning. CspE has 69% amino acid sequence identity with the major cold shock protein in E. coli, CspA, which has been shown to bind to a DNA sequence designated the Y box, with the sequence 5'-ATTGG. Of the nine templates tested, CspE was found to be most heavily crosslinked to RNA from the lambda PR' promoter, which is modified by the Q antiterminator protein. CspE was very heavily crosslinked to RNA only ten nucleotides long in initial ternary complexes on this promoter, but not to this same RNA after it had been released from the transcription complex. However, even when present from the start of transcription, CspE did not crosslink to the RNA 82 nucleotides long in elongation complexes from this same promoter. Despite the loss of interaction with the RNA after polymerase had left the promoter, CspE inhibited Q-mediated transcriptional antitermination from PR' in vitro almost 200 nucleotides downstream from the promoter, presumably by interaction with the Y box DNA upstream from PR', which overlaps with the binding site for the Q. A potential role for CspE and transcription in chromosome condensation and nucleoid structure is discussed.
利用光交联核糖核苷酸类似物5-(叠氮苯甲酰)硫代-UTP或5-(叠氮苯甲酰)硫代-CTP作为转录底物，检测了部分分级分离的大肠杆菌提取物中与来自多个启动子的活性转录复合物中的新生RNA相互作用的蛋白质。在用三元转录复合物进行照射后，几种提取物蛋白质与RNA发生了交联。最值得注意的是，在测试的九个模板中的七个上，一种小蛋白质与复合物中的RNA发生了交联。这种蛋白质被纯化并测序，发现与一种假设的蛋白质MsmC/CspE匹配，最近显示该蛋白质参与染色质分配。CspE与大肠杆菌中的主要冷休克蛋白CspA具有69%的氨基酸序列同一性，CspA已被证明可与一个名为Y盒的DNA序列结合，其序列为5'-ATTGG。在测试的九个模板中，发现CspE与来自λPR'启动子的RNA交联程度最高，该启动子被Q抗终止蛋白修饰。在这个启动子上的初始三元复合物中，CspE与仅10个核苷酸长的RNA非常强烈地交联，但在其从转录复合物释放后，不与相同的RNA交联。然而，即使从转录开始就存在，CspE也不会与来自同一启动子的延伸复合物中82个核苷酸长的RNA交联。尽管在聚合酶离开启动子后与RNA的相互作用丧失，但CspE在体外抑制了从PR'下游近200个核苷酸处的Q介导的转录抗终止，推测是通过与PR'上游的Y盒DNA相互作用，该区域与Q的结合位点重叠。讨论了CspE和转录在染色体凝聚和类核结构中的潜在作用。
