Cleavage Mediated by the Catalytic Domain of Bacterial RNase P RNA Shiying Wu, Ema Kikovska, Magnus Lindell and Leif A. Kirsebom⁎ Department of Cell and Molecular Biology, Box 596, Biomedical Centre, SE-751 24 Uppsala, Sweden Received 29 October 2011; received in revised form 11 May 2012; accepted 15 May 2012 Available online 22 May 2012 Edited by D. E. Draper Keywords: RNase P; ribozyme; divalent metal ions; tRNA precursors; tRNA processing Like other RNA molecules, RNase P RNA (RPR) is composed of domains, and these have different functions. Here, we provide data demonstrating that the catalytic (C) domain of Escherichia coli (Eco) RPR when separated from the specificity (S) domain mediates cleavage using various model RNA hairpin loop substrates. Compared to full-length Eco RPR, the rate constant, k obs , of cleavage for the truncated RPR (CP RPR) was reduced 30- to 13,000-fold depending on substrate. Specifically, the structural architec- ture of the - 1/+ 73 played a significant role where a C - 1 /G + 73 pair had the most dramatic effect on k obs . Substitution of A 248 (E. coli numbering), positioned near the cleavage site in the RNase P–substrate complex, with G in the CP RPR resulted in 30-fold improvement in rate. In contrast, strengthening the interaction between the RPR and the 3′ end of the substrate only had a modest effect. Interestingly, although deleting the S- domain gave a reduction in the rate, it resulted in a less erroneous RPR with respect to cleavage site selection. These data support and extend our understanding of the coupling between the distal interaction between the S- domain and events at the active site. Our findings will also be discussed with respect to the structure of RPR derived from different organisms. © 2012 Elsevier Ltd. All rights reserved. Introduction The endoribonuclease RNase P is involved in the processing of tRNA transcripts. Bacterial RNase P consists of one protein and one RNA subunit, referred to as C5 and RNase P RNA (RPR), respectively, while archaeal and eukaryal RNase P contain one RPR and several proteins. 1–3 Irrespec- tive of origin, however, the catalytic activity resides in the RPR, which can cleave various substrates in the absence of protein at high ionic strength. 3–7 However, RNase P activities based solely on protein have recently been reported. 8,9 The RPR can be divided into two major domains, the specificity (S) and the catalytic (C) domains. 10 Deleting the S-domain in bacterial RPR results in an RNA that can promote inefficient cleav- age of precursor tRNAs both with and without the protein. 11–13 The S-domain interacts with the T-stem/loop (TSL) region of a precursor tRNA and the TSL binding site is referred to as TBS (for recent reviews, see Refs. 1, 2, and 14). A productive interaction between TSL and TBS results in efficient and correct cleavage while a distortion or absence of this interaction can affect cleavage efficiency and site selection. 7,15,16 Cleavage with Escherichia coli (Eco) RPR of substrates that do not interact (or interact differently) with the TBS requires higher concentrations of Mg 2+ for optimal cleavage compared to substrates that bind produc- tively to TBS. 15,16 One model states that a productive TSL/TBS interaction influences the positioning of chemical groups and/or Mg 2+ at the cleavage *Corresponding author. E-mail address: Leif.Kirsebom@icm.uu.se. Present addresses: E. Kikovska, Alkaloid AD, R&D Department, Bul. “Aleksandar Makedonski” 12, 1000 Skopje, Republic of Macedonia; M. Lindell, Bioimics AB, Sandstensvägen 7, SE 752 67 Uppsala, Sweden. Abbreviations used: RPR, RNase P RNA; TSL, T-stem/ loop; TBS, TSL binding site; WC, Watson–Crick. doi:10.1016/j.jmb.2012.05.020 J. Mol. Biol. (2012) 422, 204–214 Contents lists available at www.sciencedirect.com Journal of Molecular Biology journal homepage: http://ees.elsevier.com.jmb 0022-2836/$ - see front matter © 2012 Elsevier Ltd. All rights reserved.