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A single mutation attenuates both the transcription termination and RNA-dependent RNA polymerase activity of T7 RNA polymerase
bioRxiv - Molecular Biology Pub Date : 2021-01-12 , DOI: 10.1101/2021.01.11.426313
Hui Wu , Ting Wei , Rui Cheng , Fengtao Huang , Xuelin Lu , Yan Yan , Bingbing Yu , Xionglue Wang , Chenli Liu , Bin Zhu

Transcription termination is one of the least understood processes of gene expression. As the prototype model for transcription studies, the single-subunit T7 RNA polymerase (RNAP) was known to response to two types of termination signals, while the mechanism underlying such termination especially the specific elements of the polymerase involved in is still unclear, due to the lack of a termination complex structure. Here we applied phage-assisted continuous evolution to obtain variants of T7 RNAP that can bypass the typical class I T7 terminator with stem-loop structure. Through in vivo selection and in vitro characterization, we discovered a single mutation S43Y that significantly decreased the termination efficiency of T7 RNAP at all transcription terminators tested. Coincidently, the S43Y mutation almost eliminates the RNA-dependent RNA polymerase (RdRp) of T7 RNAP without affecting the major DNA-dependent RNA polymerase (DdRp) activity of the enzyme, indicating the relationship between transcription termination and RdRp activity, and suggesting a model in which the stem-loop terminator induces the RdRp activity which competes with the ongoing DdRp activity to cause transcription termination. The T7 RNAP S43Y mutant as an enzymatic reagent for in vitro transcription reduces the undesired termination in run-off RNA synthesis and produces RNA with higher terminal homogeneity.

中文翻译:

单个突变减弱T7 RNA聚合酶的转录终止和RNA依赖性RNA聚合酶活性

转录终止是人们对基因表达缺乏了解的过程之一。作为转录研究的原型模型,已知单亚基T7 RNA聚合酶(RNAP)对两种类型的终止信号有反应,而由于这种终止的机制,尤其是所涉及的聚合酶的特定元件,目前尚不清楚缺少终端复杂结构。在这里,我们应用噬菌体辅助的连续进化,以获得可以绕过具有茎环结构的典型I类T7终止子的T7 RNAP变体。通过体内选择和体外表征,我们发现了一个单个突变S43Y,该突变显着降低了在所有测试的转录终止子处T7 RNAP的终止效率。巧合的是,S43Y突变几乎消除了T7 RNAP的RNA依赖性RNA聚合酶(RdRp),而不会影响该酶的主要DNA依赖性RNA聚合酶(DdRp)活性,表明转录终止与RdRp活性之间的关系,并提出了一个模型,其中茎环终止子诱导RdRp活性,后者与正在进行的DdRp活性竞争而导致转录终止。T7 RNAP S43Y突变体作为一种用于体外转录的酶试剂,减少了径流RNA合成中不希望的终止,并产生了具有更高末端均一性的RNA。并提出了一种模型,其中茎环终止子诱导RdRp活性,RdRp活性与正在进行的DdRp活性竞争以引起转录终止。T7 RNAP S43Y突变体作为一种用于体外转录的酶试剂,减少了径流RNA合成中不希望的终止,并产生了具有更高末端均一性的RNA。并提出了一种模型,其中茎环终止子诱导RdRp活性,RdRp活性与正在进行的DdRp活性竞争以引起转录终止。T7 RNAP S43Y突变体作为一种用于体外转录的酶试剂,减少了径流RNA合成中不希望的终止,并产生了具有更高末端均一性的RNA。
更新日期:2021-01-13
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