Wilson K S, Conant C R, von Hippel P H
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR, 97403, USA.
J Mol Biol. 1999 Jun 25;289(5):1179-94. doi: 10.1006/jmbi.1999.2814.
We use a synthetic "primed bubble-duplex" model elongation complex developed previously to examine certain structural and thermodynamic features of the transcription elongation complex of Escherichia coli. The nucleic acid framework of this model complex consists of a linear base-paired DNA molecule with a central "bubble" of non-complementary nucleotide residues, together with a single-stranded RNA molecule that is complementary (at its 3'-end) to three to 12 nucleotide residues of one of the DNA strands within the bubble. RNA polymerase is added to this framework in trans, and on addition of rNTPs the resulting complex can elongate the 3'-end of the RNA primer in a template-dependent manner with functional properties that are indistinguishable from those of a "natural" promoter-initiated transcription elongation complex operating under the same conditions. In this study we use this model system to show that the formation of a stable elongation complex at any particular template position can be treated as an equilibrium process, and that semi-quantitative dissociation constants can be estimated for the complex by using a gel band-shift assay to monitor the binding of the RNA oligomer to the complex. We then show that the formation of a stable complex depends on the presence of a complementary RNA-DNA hybrid that is at least 9 bp in length, and in addition that several nucleotide residues of non-complementary RNA located upstream of the RNA-DNA hybrid bind strongly to the putative single-stranded RNA binding site of the polymerase and significantly enhance the stability of the resulting elongation complex. Finally, we demonstrate that the measured stabilities of the model constructs in which the length of the RNA-DNA hybrid is varied correlate well with the transcriptional processivity of the functioning complex that results when rNTPs are added. These findings are discussed in the context of related studies of both model systems and natural elongation complexes. The general concepts that emerge are used to define some central structural and functional features of the transcription complex.
我们使用先前开发的一种合成“引发气泡双链体”模型延伸复合物，来研究大肠杆菌转录延伸复合物的某些结构和热力学特征。该模型复合物的核酸框架由一个线性碱基配对的DNA分子组成，其中央有一个非互补核苷酸残基的“气泡”，以及一个单链RNA分子，该RNA分子（在其3'端）与气泡内一条DNA链的三到十二个核苷酸残基互补。RNA聚合酶以反式添加到这个框架上，加入核糖核苷三磷酸（rNTPs）后，所得复合物能够以模板依赖的方式延伸RNA引物的3'端，其功能特性与在相同条件下运行的“天然”启动子起始的转录延伸复合物无法区分。在本研究中，我们使用这个模型系统来表明，在任何特定模板位置形成稳定的延伸复合物都可以被视为一个平衡过程，并且可以通过使用凝胶带移分析法监测RNA寡聚物与复合物的结合来估计该复合物的半定量解离常数。然后我们表明，稳定复合物的形成取决于至少9个碱基对长度的互补RNA-DNA杂交体的存在，此外，位于RNA-DNA杂交体上游的几个非互补RNA核苷酸残基与聚合酶假定的单链RNA结合位点强烈结合，并显著增强所得延伸复合物的稳定性。最后，我们证明，在RNA-DNA杂交体长度不同的模型构建体中测得的稳定性，与加入rNTPs时产生的功能复合物的转录持续性密切相关。在模型系统和天然延伸复合物的相关研究背景下讨论了这些发现。所出现的一般概念被用于定义转录复合物的一些核心结构和功能特征。
