TERMINATION AND SLIPPAGE BY BACTERIOPHAGE-T7 RNA-POLYMERASE
TERMINATION AND SLIPPAGE BY BACTERIOPHAGE-T7 RNA-POLYMERASE
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DOI：
10.1006/jmbi.1993.1458
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发表时间：
1993-08-20
影响因子：
5.6
通讯作者：
MCALLISTER, WT
中科院分区：
文献类型：
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作者：
MACDONALD, LE;ZHOU, YW;MCALLISTER, WT
我们研究了T7和T3 RNA聚合酶（RNAP）在各种终止信号下的终止效率。与以前的研究者一致，我们发现终止发生在具有稳定二级结构的RNA产物合成后，随后是一系列U残基。缺乏3′ U-束的茎环结构不能终止噬菌体酶。的距离转录起始位点和终止信号之间的终止子（或序列）也可能是重要的，因为将终止子放置在启动子下游的不同位置，或改变启动子序列，导致终止效率的改变。并且已经观察到当合成富含U的转录物而不是富含A或C的转录物时，酶终止（无效）。这在低浓度的UTP下尤其如此。引人注目的是，当延伸复合物（EC）遇到模板链中的dA-片段时，它能够在模板上滑动，导致合成的产物具有比DNA序列预测的更多或更少的U残基。这一观察结果表明，RNA：DNA杂交的形成可能是重要的横向稳定性的EC（其能力，以保持适当的注册与DNA模板）。我们还探讨了终止性质的蛋白水解切口形式的T7 RNAP。切口酶形成比完整RNAP更不稳定的EC，并且在编码固有不稳定RNA的区域（例如茎环结构、聚（U）-束）中更容易从模板解离。然而，切口酶在晚期T7终止子（T7-TΦ）或人前甲状旁腺激素原基因的终止信号处有效终止。这些结果表明，终止是一个高度特异性的事件，而不仅仅是一个后果的稳定性下降的EC。我们的意见是不一致的，与以前的模型终止噬菌体RNAP，并表明这些模型可能需要修改。
We have examined the termination efficiency of T7 and T3 RNA polymerases (RNAPs) at a variety of termination signals. In agreement with previous investigators we find that termination occurs after the synthesis of an RNA product with a stable secondary structure followed by a run of U residues. Stem-loop structures that lack a 3′ U-tract fail to terminate the phage enzyme. The distance (or the sequence) between the start site for transcription and the termination signal may also be important, as placing the terminator at different locations downstream from the promoter, or changing the promoter sequence, results in alterations in termination efficiency.We have explored termination at extended runs of homopolymers in the absence of an apparent stem-loop structure, and have observed that the enzyme terminates (inefficiently) when synthesizing U-rich transcripts, but not A- or C-rich transcripts. This is especially true at low concentrations of UTP. Strikingly, when an elongation complex (EC) encounters a dA-tract in the template strand it is able to slide on the template, resulting in the synthesis of products that have more or fewer U residues than predicted by the sequence of the DNA. This observation suggests that the formation of an RNA : DNA hybrid may be important to the lateral stability of the EC (its ability to maintain proper register with the DNA template).We have also explored the termination properties of a proteolytically nicked form of T7 RNAP. The nicked enzyme forms a less stable EC than the intact RNAP and dissociates more readily from the template in regions that encode inherently destabilizing RNAs (e.g. stem-loop structures, poly(U)-tracts). However, the nicked enzyme terminateslessefficiently at the late T7 terminator (T7-TΦ) or at a termination signal in the human preproparathyroid hormone gene. These results suggest that termination is a highly specific event, and not merely a consequence of decreased stability of the EC.Our observations are not consistent with previous models of termination by the phage RNAP and indicate that revisions to these models may be required.