https://scholars.lib.ntu.edu.tw/handle/123456789/416745
標題: | Functional reconstitution and characterization of Pyrococcus furiosus RNase P | 作者: | HSIN-YUE TSAI Pulukkunat, D.K. Woznick, W.K. Gopalan, V. |
關鍵字: | archaeal RNase P; in vitro reconstitution; precursor tRNA processing | 公開日期: | 2006 | 出版社: | NATL ACAD SCIENCES | 卷: | 103 | 期: | 44 | 起(迄)頁: | 16147 | 來源出版物: | Proceedings of the National Academy of Sciences of the United States of America | 摘要: | RNase P, which catalyzes the magnesium-dependent 5′-end maturation of tRNAs in all three domains of life, is composed of one essential RNA and a varying number of protein subunits depending on the source: at least one in bacteria, four in archaea, and nine in eukarya. To address why multiple protein subunits are needed for archaeal eukaryal RNase P catalysis, in contrast to their bacterial relative, in vitro reconstitution of these holoenzymes is a prerequisite. Using recombinant subunits, we have reconstituted in vitro the RNase P holoenzyme from the thermophilic archaeon Pyroccocus furiosus (Pfu) and furthered our understanding regarding its functional organization and assembly pathway(s). Whereas Pfu RNase P RNA (RPR) alone is capable of multiple turnover, addition of all four RNase P protein (Rpp) subunits to Pfu RPR results in a 25-fold increase in its kcat and a 170-fold decrease in K m. In fact, even in the presence of only one of two specific pairs of Rpps, the RPR displays activity at lower substrate and magnesium concentrations. Moreover, a pared-down, mini-Pfu RNase P was identified with an RPR deletion mutant. Results from our kinetic and foot-printing studies on Pfu RNase P, together with insights from recent structures of bacterial RPRs, provide a framework for appreciating the role of multiple Rpps in archaeal RNase P. © 2006 by The National Academy of Sciences of the USA. |
URI: | http://www.scopus.com/inward/record.url?eid=2-s2.0-33750831489&partnerID=MN8TOARS https://scholars.lib.ntu.edu.tw/handle/123456789/416745 |
ISSN: | 0027-8424 1091-6490 |
DOI: | 10.1073/pnas.0608000103 18887870 |
顯示於: | 分子醫學研究所 |
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