Transcription arrest caused by long nascent RNA chains
Transcription arrest caused by long nascent RNA chains
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DOI：
10.1016/j.bbaexp.2004.12.006
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发表时间：
2005-02-14
期刊：
影响因子：
--
通讯作者：
Nielsen, PE
中科院分区：
文献类型：
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作者：
Bentin, T;Cherny, D;Nielsen, PE
转录过程是高度进行性的。然而，在新生RNA中编码的特定序列元件可以发出转录暂停和/或终止的信号。我们发现，在一定条件下，新生的RNA链可以有一个强大的和明显的序列无关的转录抑制作用。使用噬菌体T3 RNA聚合酶（T3 RNAP）和不含任何强终止信号的共价闭合环状（cccDNA）DNA模板，转录在短时间内被严重抑制。孵育10分钟后，剩余的转录活性低于10%。RNA酶A的添加几乎完全恢复转录的剂量依赖性的方式。在RNA酶A拯救的整个过程中，延伸速率保持在170 nt/s左右，并且该速率不依赖于RNA转录物的长度，至少可达6 kb。相反，RNase A拯救增加了活性延伸复合物的数量。因此，转录表现为一个全有或全无的过程。通过电镜和生化实验探讨了转录抑制的机制。数据表明，多种机制可能有助于观察到的效果。部分抑制可以归因于新生RNA和DNA模板之间的R环的形成，其为尾随T3 RNAP提供了“路障”。基于现有的文献，我们讨论了可能的体内影响的结果。(C)2004 Elsevier B. V.保留所有权利。
The transcription process is highly processive. However, specific sequence elements encoded in the nascent RNA may signal transcription pausing and/or termination. We find that under certain conditions nascent RNA chains can have a strong and apparently sequence-independent inhibitory effect on transcription. Using phage T3 RNA polymerase (T3 RNAP) and covalently closed circular (cccDNA) DNA templates that did not contain any strong termination signal, transcription was severely inhibited after a short period of time. Less than similar to10% residual transcriptional activity remained after 10 min of incubation. The addition of RNase A almost fully restored transcription in a dose dependent manner. Throughout RNase A rescue, an elongation rate of similar to170 nt/s was maintained and this velocity was independent of RNA transcript length, at least up to 6 kb. Instead, RNase A rescue increased the number of active elongation complexes. Thus transcription behaved as an all-or-none process. The mechanism of transcription inhibition was explored using electron microscopy and further biochemical experiments. The data suggest that multiple mechanisms may contribute to the observed effects. Part of the inhibition can be ascribed to the formation of R-loops between the nascent RNA and the DNA template, which provides "roadblocks" to trailing T3 RNAPs. Based on available literature we discuss possible in vivo implications of the results. (C) 2004 Elsevier B.V. All rights reserved.