Research Article: Critical domain interactions for type A RNase P RNA catalysis with and without the specificity domain

Date Published: March 6, 2018

Publisher: Public Library of Science

Author(s): Guanzhong Mao, Abhishek S. Srivastava, Shiying Wu, David Kosek, Magnus Lindell, Leif A. Kirsebom, Lennart Randau.


The natural trans-acting ribozyme RNase P RNA (RPR) is composed of two domains in which the catalytic (C-) domain mediates cleavage of various substrates. The C-domain alone, after removal of the second specificity (S-) domain, catalyzes this reaction as well, albeit with reduced efficiency. Here we provide experimental evidence indicating that efficient cleavage mediated by the Escherichia coli C-domain (Eco CP RPR) with and without the C5 protein likely depends on an interaction referred to as the “P6-mimic”. Moreover, the P18 helix connects the C- and S-domains between its loop and the P8 helix in the S-domain (the P8/ P18-interaction). In contrast to the “P6-mimic”, the presence of P18 does not contribute to the catalytic performance by the C-domain lacking the S-domain in cleavage of an all ribo model hairpin loop substrate while deletion or disruption of the P8/ P18-interaction in full-size RPR lowers the catalytic efficiency in cleavage of the same model hairpin loop substrate in keeping with previously reported data using precursor tRNAs. Consistent with that P18 is not required for cleavage mediated by the C-domain we show that the archaeal Pyrococcus furiosus RPR C-domain, which lacks the P18 helix, is catalytically active in trans without the S-domain and any protein. Our data also suggest that the S-domain has a larger impact on catalysis for E. coli RPR compared to P. furiosus RPR. Finally, we provide data indicating that the absence of the S-domain and P18, or the P8/ P18-interaction in full-length RPR influences the charge distribution near the cleavage site in the RPR-substrate complex to a small but reproducible extent.

Partial Text

Almost all tRNAs carry a phosphate at their 5′ ends due to the action of the endoribonuclease RNase P. Bacterial RNase P consists of one protein (C5), and one RNA subunit [1]. The composition of archaeal and eukarayal RNase P is more complex where the sole RNA subunit binds several proteins [2, 3]. Available data suggest that the catalytic activity resides in the RNA irrespective of origin, and the RNA alone can cleave various substrates in the absence of protein at high ionic strength [2, 4–7]. However, recent data demonstrate the presence of a protein only RNase P activity (PRORP), for example in human mitochondria and in Arabidopsis thaliana, that possesses the capacity to cut precursor tRNAs at the same site as RNase P [8, 9].




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