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Analysis of the cleavage mechanism by protein-only RNase P using precursor tRNA substrates with modifications at the cleavage siteстатья
Статья опубликована в высокорейтинговом журнале
Информация о цитировании статьи получена из
Web of Science,
Scopus
Статья опубликована в журнале из списка Web of Science и/или Scopus
Дата последнего поиска статьи во внешних источниках: 13 февраля 2017 г.
- Авторы: Walczyk Dennis, Gößringer Markus, Rossmanith Walter, Zatsepin Timofei S., Oretskaya Tatiana S., Hartmann Roland K.
- Журнал: Journal of Molecular Biology
- Том: 428
- Номер: 24
- Год издания: 2016
- Издательство: Academic Press
- Местоположение издательства: United States
- Первая страница: 4917
- Последняя страница: 4928
- DOI: 10.1016/j.jmb.2016.10.020
- Аннотация: Ribonuclease P (RNase P) is the enzyme that endonucleolytically removes 5′-precursor sequences from tRNA transcripts in all domains of life. RNase P activities are either ribonucleoprotein (RNP) or protein-only (PRORP) enzymes, raising the question about the mechanistic strategies utilized by these architecturally different enzyme classes to catalyze the same type of reaction. Here we analyzed the kinetics and cleavage-site selection by PRORP3 from Arabidopsis thaliana (AtPRORP3) using precursor tRNAs (pre-tRNAs) with individual modifications at the canonical cleavage site, either Rp- or Sp-phosphorothioate, or 2′-deoxy, 2′-fluoro, 2′-amino or 2′-O-methyl substitutions. We observed a small but robust rescue effect of Sp-phosphorothioate-modified pre-tRNA in the presence of thiophilic Cd2 + − ions, consistent with metal ion coordination to the (pro-)Sp-oxygen during catalysis. Sp-phosphorothioate, 2′-deoxy, 2′-amino and 2′-O-methyl modification redirected cleavage mainly to the next unmodified phosphodiester in the 5′-direction. Our findings are in line with the 2′-OH substituent at nt − 1 being involved in an H bonding acceptor function. In contrast to bacterial RNase P, AtPRORP3 was found to be able to utilize the canonical and upstream cleavage site with similar efficiency (corresponding to reduced cleavage fidelity), thus the two cleavage pathways appear less interdependent than in the bacterial RNA-based system.
- Добавил в систему: Зацепин Тимофей Сергеевич
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