In:
RNA, Cold Spring Harbor Laboratory, Vol. 22, No. 11 ( 2016-11), p. 1699-1709
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 50 of ∼2 nM) when incubated with preassembled B. subtilis RNase P holoenzymes.
Type of Medium:
Online Resource
ISSN:
1355-8382
,
1469-9001
DOI:
10.1261/rna.057422.116
Language:
English
Publisher:
Cold Spring Harbor Laboratory
Publication Date:
2016
detail.hit.zdb_id:
1475737-0
SSG:
12
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