Characterization of RNA-based and protein-only RNases P from bacteria encoding both enzyme types.

A small group of bacteria encode two types of RNase P, the classical ribonucleoprotein (RNP) RNase P as well as the protein-only RNase P HARP (homolog of Aquifex RNase P). We characterized the dual RNase P activities of five bacteria that belong to three different phyla. All five bacterial species encode functional RNA (gene rnpB) and protein (gene rnpA) subunits of RNP RNase P, but only the HARP of the thermophile Thermodesulfatator indicus (phylum Thermodesulfobacteria) was found to have robust tRNA 5'-end maturation activity in vitro and in vivo in an Escherichia coli RNase P depletion strain. These findings suggest that both types of RNase P are able to contribute to the essential tRNA 5'-end maturation activity in T. indicus, thus resembling the predicted evolutionary transition state in the progenitor of the Aquificaceae before the loss of rnpA and rnpB genes in this family of bacteria. Remarkably, T. indicus RNase P RNA is transcribed with a P12 expansion segment that is posttranscriptionally excised in vivo, such that the major fraction of the RNA is fragmented and thereby truncated by ∼70 nt in the native T. indicus host as well as in the E. coli complementation strain. Replacing the native P12 element of T. indicus RNase P RNA with the short P12 helix of Thermotoga maritima RNase P RNA abolished fragmentation, but simultaneously impaired complementation efficiency in E. coli cells, suggesting that intracellular fragmentation and truncation of T. indicus RNase P RNA may be beneficial to RNA folding and/or enzymatic activity.

RNase P Inhibitors Identified as Aggregators.

RNase P is an essential enzyme responsible for tRNA 5'-end maturation. In most bacteria, the enzyme is a ribonucleoprotein consisting of a catalytic RNA subunit and a small protein cofactor termed RnpA. Several studies have reported small-molecule inhibitors directed against bacterial RNase P that were identified by high-throughput screenings. Using the bacterial RNase P enzymes from Thermotoga maritima, Bacillus subtilis, and Staphylococcus aureus as model systems, we found that such compounds, including RNPA2000 (and its derivatives), iriginol hexaacetate, and purpurin, induce the formation of insoluble aggregates of RnpA rather than acting as specific inhibitors. In the case of RNPA2000, aggregation was induced by Mg2+ ions. These findings were deduced from solubility analyses by microscopy and high-performance liquid chromatography (HPLC), RnpA-inhibitor co-pulldown experiments, detergent addition, and RnpA titrations in enzyme activity assays. Finally, we used a B. subtilis RNase P depletion strain, whose lethal phenotype could be rescued by a protein-only RNase P of plant origin, for inhibition zone analyses on agar plates. These cell-based experiments argued against RNase P-specific inhibition of bacterial growth by RNPA2000. We were also unable to confirm the previously reported nonspecific RNase activity of S. aureus RnpA itself. Our results indicate that high-throughput screenings searching for bacterial RNase P inhibitors are prone to the identification of "false positives" that are also termed pan-assay interference compounds (PAINS).

[Modified Oligonucleotides for Guiding RNA Cleavage Using Bacterial RNase P].

The ability of a series of novel modified external guide sequences (EGS oligonucleotides) to induce the hydrolysis of target RNA with bacterial ribonuclease P has been studied; the most efficient modification variants have been selected. We have found patterns of the oligonucleotide sugar-phosphate backbone modi-fications that enhance oligonucleotide stability in the biological environment and do not violate the ability to interact with the enzyme and induce the RNA hydrolysis. It has been shown that analogues of EGS oligonucleotides selectively modified at 2'-position (2'-O-methyl and 2'-fluoro) or at internucleotide phosphates (phosphoryl guanidines) can be used for the addressed cleavage of a model RNA target by bacterial RNase P. The ability of new phosphoryl guanidine analogues of oligodeoxyribonucleotides that are stable in biological media to induce the hydrolysis of target RNA with bacterial ribonuclease P has been shown for the first time. The modified EGS oligonucleotides with an optimal balance between functional activity and stability in biological media can be considered as potential antibacterial agents.

Novel Peptide Conjugates of Modified Oligonucleotides for Inhibition of Bacterial RNase P.

Novel alternatives to traditional antibiotics are now of great demand for the successful treatment of microbial infections. Here, we present the engineering and properties of new oligonucleotide inhibitors of RNase P, an essential bacterial enzyme. The series of 2'-O-methyl RNA (2'-OMe-RNA) and phosphoryl guanidine oligonucleotides were targeted to the substrate-binding region of M1 RNA subunit of the RNase P. Uniformly modified 2'-OMe RNA and selectively modified phosphoryl guanidine oligonucleotides possessed good stability in biological media and effectively inhibited RNase P. Their conjugates with transporting peptides were shown to penetrate bacterial cells (Escherichia coli and Acinetobacter baumannii) and inhibit bacterial growth.