Komissarova N, Kashlev M
Advanced BioScience Laboratories-Basic Research Program, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702-1201, USA.
Proc Natl Acad Sci U S A. 1998 Dec 8;95(25):14699-704. doi: 10.1073/pnas.95.25.14699.
To determine the dynamics of transcript extrusion from Escherichia coli RNA polymerase (RNAP), we used degradation of the RNA by RNases T1 and A in a series of consecutive elongation complexes (ECs). In intact ECs, even extremely high doses of the RNases were unable to cut the RNA closer than 14-16 nt from the 3' end. Our results prove that all of the cuts detected within the 14-nt zone are derived from the EC that is denatured during inactivation of the RNases. The protected zone monotonously translocates along the RNA after addition of new nucleotides to the transcript. The upstream region of the RNA heading toward the 5' end is cleaved and dissociated from the EC, with no effect on the stability and activity of the EC. Most of the current data suggest that an 8- to 10-nt RNA.DNA hybrid is formed in the EC. Here, we show that an 8- to 10-nt RNA obtained by truncating the RNase-generated products further with either GreB or pyrophosphate is sufficient for the high stability and activity of the EC. This result suggests that the transcript-RNAP interaction that is required for holding the EC together can be limited to the RNA region involved in the 8- to 10-nt RNA.DNA hybrid.
为了确定转录本从大肠杆菌RNA聚合酶（RNAP）挤出的动力学过程，我们在一系列连续延伸复合物（ECs）中利用核糖核酸酶T1和A对RNA进行降解。在完整的ECs中，即使是极高剂量的核糖核酸酶也无法切割距3'端小于14 - 16个核苷酸的RNA。我们的结果证明，在14个核苷酸区域内检测到的所有切割都源自核糖核酸酶失活过程中变性的EC。在向转录本添加新核苷酸后，保护区沿着RNA单调移动。朝向5'端的RNA上游区域被切割并与EC解离，而对EC的稳定性和活性没有影响。目前大多数数据表明，在EC中会形成8至10个核苷酸的RNA - DNA杂交体。在此，我们表明，通过用GreB或焦磷酸进一步截短核糖核酸酶产生的产物而获得的8至10个核苷酸的RNA足以维持EC的高稳定性和活性。这一结果表明，将EC结合在一起所需的转录本 - RNAP相互作用可能仅限于参与8至10个核苷酸RNA - DNA杂交体的RNA区域。
