Bacterial type B RNase P: functional characterization of the L5.1-L15.1 tertiary contact and antisense inhibition.
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 (IC50of ∼2 nM) when incubated with preassembled B. subtilis RNase P holoenzymes.
Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, D-35037 Marburg, Germany.
Dennis
D
Walczyk
Klinik für Infektiologie und Mikrobiologie, Universitätsklinikum Schleswig-Holstein Campus Lübeck, D-23538 Lübeck, Germany.
Dagmar K
DK
Willkomm
Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, D-35037 Marburg, Germany roland.hartmann@staff.uni-marburg.de.
Roland K
RK
Hartmann
D001412:Bacillus subtilis / Q000201:enzymology*; D020134:Catalytic Domain; D005816:Genetic Complementation Test; D007700:Kinetics; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012329:RNA, Bacterial / Q000737:chemistry; D043262:Ribonuclease P / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D016376:Oligonucleotides, Antisense; D012329:RNA, Bacterial; D043262:Ribonuclease P
B. subtilis P RNA; antisense inhibition; kinetics; mutation of L5.1-L15.1 contact; structure probing