T7 RNA聚合酶
RNA聚合酶
生物
终端(太阳能)
抄写(语言学)
RNA依赖性RNA聚合酶
终止因子
聚合酶
核糖核酸
反终止
RNA聚合酶Ⅱ
分子生物学
一般转录因子
细胞生物学
DNA
遗传学
基因表达
基因
噬菌体
发起人
大肠杆菌
电离层
物理
哲学
语言学
天文
作者
Hui Wu,Ting Wei,Bingbing Yu,Rui Cheng,Fengtao Huang,Xuelin Lu,Yan Yan,Xionglue Wang,Chenli Liu,Bin Zhu
出处
期刊:RNA Biology
[Informa]
日期:2021-07-27
卷期号:18 (sup1): 451-466
被引量:13
标识
DOI:10.1080/15476286.2021.1954808
摘要
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) is known to respond to two types of termination signals, but the mechanism underlying such termination, especially the specific elements of the polymerase involved, is still unclear, due to a lack of knowledge with respect to the structure of the termination complex. 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 RNAP (RdRp) activity of T7 RNAP without impeding the major DNA-dependent RNAP (DdRp) activity of the enzyme. S43 is located in a hinge region and regulates the transformation between transcription initiation and elongation of T7 RNAP. Steady-state kinetics analysis and an RNA binding assay indicate that the S43Y mutation increases the transcription efficiency while weakening RNA binding of the enzyme. As an enzymatic reagent for in vitro transcription, the T7 RNAP S43Y mutant reduces the undesired termination in run-off RNA synthesis and produces RNA with higher terminal homogeneity.
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