单亚基RNA聚合酶,KpnP, Ro45Iw和CD23823,具有精确的末端合成。

IF 4 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Haruka Takatsuki, Ryota Miyachi, Kaito Seo, Katsumi Hagino, Norikazu Ichihashi
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引用次数: 0

摘要

T7 RNA聚合酶(RNAP)是一种高活性的单肽聚合酶,广泛用于体外RNA合成。T7 RNAP合成具有异质3′端的RNA,有时会产生问题。迄今为止,已经报道了一些rnap(例如KP34和Syn5)合成具有更精确的3'端RNA。但它们有自己的特点,不能完全替代T7 RNAP。为了增加可用RNAP库的数量,我们在噬菌体RNAP数据库中搜索其他RNAP及其启动子。从第一次筛选的9个rnap中,我们选择了3个rnap,分别命名为KpnP、Ro45Iw和CD23823,以及它们的启动子序列。重组rnap的表征表明,与T7相比,这三种聚合酶具有相似的RNA合成活性,但偏好稍低的温度。此外,CD23823表现出稍高的耐盐性。对合成RNA的5‘和3’末端进行深度测序发现,这三种RNAP,尤其是CD23823,比T7 RNAP产生更均匀的末端。这些结果表明,这些以前未被表征的rnap,特别是CD23823,对于需要精确的5‘和3’端序列的RNA合成是有用的。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Single-subunit RNA polymerases, KpnP, Ro45Iw, and CD23823, with precise terminal synthesis.

T7 RNA polymerase (RNAP) is a highly active single-peptide polymerase that is widely used for in vitro RNA synthesis. T7 RNAP synthesizes RNA with a heterogeneous 3'-end, which sometimes causes problems. To date, some RNAPs (e.g., KP34 and Syn5) that synthesize RNA with more precise 3'-ends have been reported. However, they have their own characteristics and thus do not fully substitute T7 RNAP. To increase the number of usable RNAP repertoires, we searched for other RNAPs and their promoters in the phage RNAP database. From the first screening of nine RNAPs, we selected three RNAPs, named KpnP, Ro45Iw, and CD23823, together with their promoter sequences. Characterization of recombinant RNAPs revealed that compared to T7, these three polymerases exhibited similar RNA synthesis activities but preferred a slightly lower temperature. Additionally, CD23823 exhibited a slightly higher salt tolerance. Deep sequencing of the 5'- and 3'-termini of the synthesized RNA revealed that the three RNAPs, particularly CD23823, produced more homogeneous termini than T7 RNAP. These results indicate that these previously uncharacterized RNAPs, especially CD23823, are useful for RNA synthesis that requires precise 5'- and 3'-end sequences.

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来源期刊
Journal of Biological Chemistry
Journal of Biological Chemistry Biochemistry, Genetics and Molecular Biology-Biochemistry
自引率
4.20%
发文量
1233
期刊介绍: The Journal of Biological Chemistry welcomes high-quality science that seeks to elucidate the molecular and cellular basis of biological processes. Papers published in JBC can therefore fall under the umbrellas of not only biological chemistry, chemical biology, or biochemistry, but also allied disciplines such as biophysics, systems biology, RNA biology, immunology, microbiology, neurobiology, epigenetics, computational biology, ’omics, and many more. The outcome of our focus on papers that contribute novel and important mechanistic insights, rather than on a particular topic area, is that JBC is truly a melting pot for scientists across disciplines. In addition, JBC welcomes papers that describe methods that will help scientists push their biochemical inquiries forward and resources that will be of use to the research community.
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