Kireeva M L, Komissarova N, Kashlev M
ABL-Basic Research Program, NCI - Frederick Cancer Research and Development Center, Bldg. 539, Room 222, Frederick, MD, 21702-1201, USA.
J Mol Biol. 2000 Jun 2;299(2):325-35. doi: 10.1006/jmbi.2000.3755.
An eight nucleotide RNA:DNA hybrid at the 3' end of the transcript is required for the stability of the elongation complex (EC) of RNA polymerase II. A non-template DNA strand is not needed for the stability of the EC, which contains this minimal hybrid. Here, we apply a recently developed method for promoter-independent assembly of functional EC of RNA polymerase II from synthetic RNA and DNA oligonucleotides to study the minimal composition of the nucleic acid array required for stability of the complex with RNA longer than eight nucleotides. We found that upon RNA extension beyond 14-16 nt in the course of transcription, non-template DNA becomes essential for maintaining a stable EC. Our data suggest that the overextended RNA:DNA hybrid formed in the absence the non-template DNA acts as a negative regulator of EC stability. The dissociation of the EC correlates with the backsliding of the polymerase along the overextended hybrid. The dual role of the hybrid provides a mechanism for the control of a correct nucleic acid architecture in the EC and of RNA polymerase II processivity.
转录本3'端的一个八核苷酸RNA:DNA杂交体对于RNA聚合酶II延伸复合物（EC）的稳定性是必需的。对于包含这种最小杂交体的EC的稳定性而言，非模板DNA链并非必需。在此，我们应用一种最近开发的方法，从合成的RNA和DNA寡核苷酸中进行RNA聚合酶II功能性EC的无启动子组装，以研究对于与长度超过八个核苷酸的RNA形成稳定复合物而言所需的核酸阵列的最小组成。我们发现，在转录过程中当RNA延伸超过14 - 16个核苷酸时，非模板DNA对于维持稳定的EC变得至关重要。我们的数据表明，在不存在非模板DNA的情况下形成的过度延伸的RNA:DNA杂交体充当EC稳定性的负调节因子。EC的解离与聚合酶沿着过度延伸的杂交体的后滑相关。这种杂交体的双重作用为控制EC中正确的核酸结构以及RNA聚合酶II的持续合成能力提供了一种机制。
