Dissinger S, Hanna M M
Department of Biological Chemistry, California College of Medicine, University of California, Irvine 92717.
J Mol Biol. 1991 May 5;219(1):11-25. doi: 10.1016/0022-2836(91)90853-x.
We have isolated Escherichia coli transcription complexes, paused in the presence and absence of Nus A, which contain RNA substituted at every UMP residue with a photocrosslinking nucleotide analog. The pause site is immediately downstream from an RNA stem-loop structure, and although pausing occurs in the absence of Nus A, it is substantially enhanced in the presence of Nus A. We have analyzed the secondary structure of this RNA and show that the analog does not interfere with the formation of the normal stem-loop structures. Additionally, the analog substrate does not alter transcriptional pausing, in the presence or absence of Nus A, indicating that Nus A recognition of the transcription complex is not affected by the presence of the crosslinking groups in the RNA. Ribonuclease digestion of the RNA in paused complexes identifies two accessible regions, two nucleotides in the loop and one near the base of the upstream side of the stem-loop. Cleavage at one loop nucleotide is enhanced by Nus A, while the nucleotide near the base of the stem-loop is partially protected. Upon irradiation of the transcription complex, Nus A is not photoaffinity labeled by the RNA, even at a high molar ration to RNA polymerase (250:1). Both the beta and beta' subunits are labeled, however, indicating that the putative stem-loop binding domain on the core polymerase involves both subunits. Because the nucleotide protected from ribonuclease by Nus A is very near two analogs, yet Nus A is not crosslinked to the RNA, it is unlikely that Nus A could be protecting this position through direct contact. Furthermore, analog is substituted at positions in both the loop and at several positions in the stem, and again, no crosslinking to Nus A is observed. We conclude that enhancement of pausing by Nus A probably does not require direct interaction with the bases in the RNA stem-loop.
我们分离出了大肠杆菌转录复合物，这些复合物在有和没有Nus A的情况下都会暂停，其中的RNA在每个UMP残基处都被光交联核苷酸类似物取代。暂停位点紧挨着一个RNA茎环结构的下游，虽然在没有Nus A的情况下也会发生暂停，但在有Nus A的情况下会显著增强。我们分析了该RNA的二级结构，结果表明类似物不会干扰正常茎环结构的形成。此外，无论有无Nus A，类似物底物都不会改变转录暂停，这表明RNA中交联基团的存在不会影响Nus A对转录复合物的识别。对暂停复合物中的RNA进行核糖核酸酶消化，可确定两个可及区域，一个是环中的两个核苷酸，另一个靠近茎环上游一侧的基部。Nus A会增强环中一个核苷酸处的切割，而茎环基部附近的核苷酸则受到部分保护。照射转录复合物后，即使Nus A与RNA聚合酶的摩尔比很高（250:1），Nus A也不会被RNA光亲和标记。然而，β和β'亚基都被标记了，这表明核心聚合酶上假定的茎环结合结构域涉及这两个亚基。由于被Nus A保护免受核糖核酸酶作用的核苷酸非常靠近两个类似物，但Nus A并未与RNA交联，因此Nus A不太可能通过直接接触来保护这个位置。此外，类似物在环中和茎中的几个位置都有取代，同样也未观察到与Nus A的交联。我们得出结论，Nus A对暂停的增强作用可能不需要与RNA茎环中的碱基直接相互作用。
