Bacterial type B RNase P: functional characterization of the L5.1-L15.1 tertiary contact and antisense inhibition

Abstract

Ribonuclease P is the ubiquitous endonuclease that generates the mature 5'-ends of precursor tRNAs. In bacteria, the enzyme is composed of a catalytic RNA (∼400 nucleotides) and a small essential protein subunit (∼13 kDa). Most bacterial RNase P RNAs (P RNAs) belong to the architectural type A; type B RNase P RNA is confined to the low-G+C Gram-positive bacteria. Here we demonstrate that the L5.1-L15.1 intradomain contact in the catalytic domain of the prototypic type B RNase P RNA of Bacillus subtilis is crucial for adopting a compact functional conformation: Disruption of the L5.1-L15.1 contact by antisense oligonucleotides or mutation reduced P RNA-alone and holoenzyme activity by one to two orders of magnitude in vitro, largely retarded gel mobility of the RNA and further affected the structure of regions P7/P8/P10.1, P15 and L15.2, and abolished the ability of B. subtilis P RNA to complement a P RNA-deficient Escherichia coli strain. We also provide mutational evidence that an L9-P1 tertiary contact, as found in some Mycoplasma type B RNAs, is not formed in canonical type B RNAs as represented by B. subtilis P RNA. We finally explored the P5.1 and P15 stem-loop structures as targets for LNA-modified antisense oligonucleotides. Oligonucleotides targeting P15, but not those directed against P5.1, were found to efficiently anneal to P RNA and to inhibit activity (IC of ∼2 nM) when incubated with preassembled B. subtilis RNase P holoenzymes.

Homologs of aquifex aeolicus protein‐only RNase P are not the major RNase P activities in the archaea haloferax volcanii and methanosarcina mazei

“…(A) Secondary structure of the class I pre‐tRNA Gly from the thermophilic bacterium Thermus thermophilus carrying a 14‐nt 5′‐leader. The canonical RNase P cleavage site (between nt −1 and + 1, termed c 0 site; is indicated. (B) RNase P processing of 5′‐ 32 P‐labeled T. thermophilus pre‐tRNA Gly by recombinant HARPs from the hyperthermophilic bacterium A. aeolicus (Aq_880cHis), the halophilic archaeon H. volcanii (Hvo_1114cHis) and the strictly anaerobic methanogenic archaeon M. mazei (Mm_2077nHis).…”

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

“…This is in contrast to the 120-fold reduction in the rate observed for the type A Eco RPR ( Table 1 ; see also Refs [ 22 , 23 ]). Type B lacks P6, however, recent data indicate that disruption of the intra-domain interaction between L5.1 and L15.1 in the C-domain affects both folding and the catalytic activity in a full-size RPR context [ 27 ]. Hence, it will be of interest to understand whether the L5.1/ L15.1 interaction (or a mimic) is present in the absence of the S-domain (see also Ref [ 21 ]).…”

“…However, Pfu RPR lacks P18 and consequently the P8/ 18-interaction is missing [ 6 ]. The P6- and P8/ 18-interactions are absent in type B RPR, however, the intra-domain contact L5.1/L15.1 may fulfill the role of P6 in type B [ 10 , 27 ]. Of interest, in this context we note that type T RPRs have degenerated S-domains but modeling suggest that P6 is present while P18 is absent [ 12 , 13 ].…”