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Evidence for induced fit in bacterial RNase P RNA-mediated cleavage
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
2007 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 372, no 5, 1149-1164 p.Article in journal (Refereed) Published
Abstract [en]

RNase P with its catalytic RNA subunit is involved in the processing of a number of RNA precursors with different structures. However, precursor tRNAs are the most abundant substrates for RNase P. Available data suggest that a tRNA is folded into its characteristic structure already at the precursor state and that RNase P recognizes this structure. The tRNA D-/T-loop domain (TSL-region) is suggested to interact with the specificity domain of RNase P RNA while residues in the catalytic domain interact with the cleavage site. Here, we have studied the consequences of a productive interaction between the TSL-region and its binding site (TBS) in the specificity domain using tRNA precursors and various hairpin-loop model substrates. The different substrates were analyzed with respect to cleavage site recognition, ground-state binding, cleavage as a function of the concentration of Mg2+ and the rate of cleavage under conditions where chemistry is suggested to be rate limiting using wild-type Escherichia coli RNase P RNA, M1 RNA, and M1 RNA variants with structural changes in the TBS-region. On the basis of our data, we conclude that a productive TSL/TBS interaction results in a conformational change in the M1 RNA substrate complex that has an effect on catalysis. Moreover, it is likely that this conformational change comprises positioning of chemical groups (and Mg2+) at and in the vicinity of the cleavage site. Hence, our findings are consistent with an induced-fit mechanism in RNase P RNA-mediated cleavage.

Place, publisher, year, edition, pages
2007. Vol. 372, no 5, 1149-1164 p.
Keyword [en]
RNase P, ribozyme, divalent metal ions, tRNA precursors, tRNA processing
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-14904DOI: 10.1016/j.jmb.2007.07.030ISI: 000249817500003PubMedID: 17719605OAI: oai:DiVA.org:uu-14904DiVA: diva2:42675
Available from: 2008-01-31 Created: 2008-01-31 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Distal to Proximal—Functional Coupling in RNase P RNA-mediated Catalysis
Open this publication in new window or tab >>Distal to Proximal—Functional Coupling in RNase P RNA-mediated Catalysis
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

RNase P is a ubiquitous ribonuclease responsible for removing the 5’ leader of tRNA precursor. Bacterial RNase P contains one RNA (RPR) and one protein (RPP) subunit. However, the number of protein variants depends on the origin. The RNA subunit is the catalytic subunit that in vitro cleaves its substrate with and without the protein subunit. Therefore RNase P is a ribozyme. However, the protein subunit is indispensable in vivo.

The objective of this thesis was to understand the mechanism of and substrate interaction in RPR-mediated cleavage, in particular the contributions of the two domains of RPR and the roles of the base at the -1 residue in the substrate. As model systems I have used bacterial (Eco) and archaeal (Pfu) RPRs.

The TSL (T-stem-loop) region of a tRNA precursor and the TBS (TSL-binding site) in the RPR S-domain interact upon RPR-substrate complex conformation. A productive TSL/TBS-interaction affects events at the cleavage site by influencing the positioning of chemical groups and/ or Mg2+ such that efficient and correct cleavage occurs consistent with an induced fit mechanism. With respect to events at the cleavage site, my data show that the identity of the residue immediately upstream the 5’ of the cleavage site (at -1) plays a significant role for efficient and accurate cleavage although its presence is not essential. My data also show that the RPR C-domain can cleave without the S-domain. However, the presence of the S-domain increases the efficiency of cleavage but lowers the accuracy. The structure of the S-domain of Pfu RPR differs from that of Eco RPR and my data suggest that the Pfu S-domain does not affect the accuracy in the same way as for Eco RPR. It also appears that the proteins that bind to the Pfu S-domain play a role in formation of a productive TSL/TBS-interaction. It is therefore possible that the proteins of Pfu RNase P have evolved to take over the role of the S-domain with respect to the interaction with the TSL-region of the substrate.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 64 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 861
Keyword
Ribozyme, RNase P, Induced fit model, tRNA progressing, Substrate interaction
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-159312 (URN)978-91-554-8175-9 (ISBN)
Public defence
2011-11-11, B42, Bio mediacal Center (BMC), Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2011-10-20 Created: 2011-09-27 Last updated: 2011-11-04Bibliographically approved

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