Fluorescence characterization of the transcription bubble in elongation complexes of T7 RNA polymerase
Fluorescence characterization of the transcription bubble in elongation complexes of T7 RNA polymerase
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DOI：
10.1006/jmbi.2001.4601
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发表时间：
2001-05-04
影响因子：
5.6
通讯作者：
Martin, CT
中科院分区：
文献类型：
--
作者：
Liu, CH;Martin, CT
转录延伸的各种动力学和热力学模型都需要了解随 RNA 聚合酶活性位点移动的熔化气泡的性质。气泡的一般性质是否依赖于系统，或者是否存在限制任何 RNA 聚合酶中气泡的常见能量需求？ T7 RNA 聚合酶是最简单的 RNA 聚合酶之一，也是我们拥有最高分辨率结构信息的系统。然而，没有可用于稳定伸长复合物的高分辨率信息。为了直接绘制功能性暂停延伸复合物中 DNA 熔解区域的图谱，我们将荧光探针位点特异性地引入到 DNA 中。与替代腺嘌呤碱基的2-氨基嘌呤一样，替代胞嘧啶碱基的新型探针吡咯-dC的荧光强度对其环境敏感。具体而言，相对于单链 DNA 中的荧光，双链 DNA 中的荧光被猝灭，使得探针提供有关 DNA 局部熔解的直接信息。将这种新探针放置在非模板链中的特定位置清楚地表明，延伸气泡在暂停位点上游延伸了约八个碱基，而2-氨基嘌呤探针表明延伸气泡仅在最后掺入的碱基下游延伸了约一个核苷酸。活性位点的定位非常靠近气泡的下游边缘，这与之前的研究以及与启动子结合的预引发复合物的类似研究一致。结果清楚地表明，RNA:DNA 杂交体的长度不能超过 8 个核苷酸，并且不同暂停物种的表征初步表明这些尺寸不依赖于序列或位置。最后，结果证实，三元复合物对于短转录本并不稳定，但当在双链 DNA 的中间或（径流）末端暂停时，会持续相当长的时间。 (C) 2001 年学术出版社。
The various kinetic and thermodynamic models for transcription elongation all require an understanding of the nature of the melted bubble which moves with the RNA polymerase active site. Is the general nature of the bubble system-dependent or are there common energetic requirements which constrain a bubble in any RNA polymerases? T7 RNA polymerase is one of the simplest RNA polymerases and is the system for which we have the highest-resolution structural information. However, there is no high-resolution information available for a stable elongation complex. In order to directly map melted regions of the DNA in a functionally paused elongation complex, we have introduced fluorescent probes site-specifically into the DNA. Like 2-aminopurine, which substitutes for adenine bases, the fluorescence intensity of the new probe, pyrrolo-dC, which substitutes for cytosine bases, is sensitive to its environment. Specifically, the fluorescence is quenched in duplex DNA relative to its fluorescence in single-stranded DNA, such that the probe provides direct information on local melting of the DNA. Placement of this new probe at specific positions in the non-template strand shows clearly that the elongation bubble extends about eight bases upstream of the pause site, while 2-aminopurine probes show that the elongation bubble extends only about one nucleotide downstream of the last base incorporated. The positioning of the active site very close to the downstream edge of the bubble is consistent with previous studies and with similar studies of the promoter-bound, pre-initiation complex. The results show clearly that the RNA:DNA hybrid can be no more than eight nucleotides in length, and characterization of different paused species suggests preliminarily that these dimensions are not sequence or position dependent. Finally, the results confirm that the ternary complex is not stable with short lengths of transcript, but persists for a substantial time when paused in the middle or at the (runoff) end of duplex DNA. (C) 2001 Academic Press.