Krummel B, Chamberlin M J
Division of Biochemistry and Molecular Biology, University of California, Berkeley 94720.
J Mol Biol. 1992 May 20;225(2):239-50. doi: 10.1016/0022-2836(92)90918-a.
The structure and properties of ternary complexes of RNA polymerase are of central importance in understanding the mechanisms of transcriptional elongation and termination, and the regulation of these primary steps in gene expression. However, there has been no systematic study of the structure and properties of such complexes along a single transcription unit. Recently, we have described the isolation of a collection of halted ternary complexes of Escherichia coli RNA polymerase bearing transcripts from 11 to 35 nucleotides in length along two different transcription units (accompanying paper). Here, we report structural studies of these complexes using DNase I footprinting. Surprisingly, nearly all of the different ternary complexes have distinctly different footprints along the two DNA strands, and the position of the footprint relative to the 3' end of the transcript also varies for most complexes. Halted complexes bearing transcripts of comparable size do not have identical footprints; hence, DNA sequence as well as transcript length plays a role in determining the size and position of the footprint. These differences in structure are consistent with our earlier findings that ternary complexes can differ considerably in stability and gel mobility. The downstream boundary of the RNA polymerase in ternary complexes does not move forward regularly as successive nucleotide residues are added to the RNA chain. In contrast, the upstream boundary moves forward more or less in concert with the movement of the 3' terminus of the transcript. These factors lead to a general compression of the overall footprint as transcription proceeds, together with a steady movement of the 3' terminus of the RNA toward the downstream boundary of the polymerase. Ultimately, after the length of the RNA transcript has increased from eight to ten nucleotides, the downstream boundary of the complex is found to move downstream along the DNA, suggesting a translocation event. We suggest that RNA chain elongation, like RNA chain initiation, may involve a saltatory process in which net translocation of the complex along the DNA occurs only after addition of a number of ribonucleotides to the RNA chain.
RNA聚合酶三元复合物的结构和性质对于理解转录延伸和终止机制以及基因表达中这些主要步骤的调控至关重要。然而，尚未有针对沿着单个转录单元的此类复合物的结构和性质进行的系统研究。最近，我们描述了分离出的一系列大肠杆菌RNA聚合酶的停滞三元复合物，这些复合物携带沿着两个不同转录单元长度为11至35个核苷酸的转录本（随附论文）。在此，我们报告使用DNase I足迹法对这些复合物进行的结构研究。令人惊讶的是，几乎所有不同的三元复合物在两条DNA链上都有明显不同的足迹，并且对于大多数复合物而言，足迹相对于转录本3'端的位置也有所不同。携带大小相当的转录本的停滞复合物没有相同的足迹；因此，DNA序列以及转录本长度在决定足迹的大小和位置方面都发挥作用。这些结构上的差异与我们早期的发现一致，即三元复合物在稳定性和凝胶迁移率方面可能有很大差异。随着连续的核苷酸残基添加到RNA链上，三元复合物中RNA聚合酶的下游边界不会有规律地向前移动。相反，上游边界或多或少与转录本3'末端的移动协同向前移动。随着转录的进行，这些因素导致总体足迹普遍压缩，同时RNA的3'末端朝着聚合酶的下游边界稳定移动。最终，在RNA转录本的长度从八个核苷酸增加到十个核苷酸后，发现复合物的下游边界沿着DNA向下游移动，这表明发生了转位事件。我们认为，RNA链延伸与RNA链起始一样，可能涉及一个跳跃过程，即复合物沿着DNA的净转位仅在向RNA链添加了多个核糖核苷酸之后才会发生。
