The previously uncharacterized RnpM (YlxR) protein modulates the activity of ribonuclease P in Bacillus subtilis in vitro

Abstract

Even though Bacillus subtilis is one of the most studied organisms, no function has been identified for about 20% of its proteins. Among these unknown proteins are several RNA- and ribosome-binding proteins suggesting that they exert functions in cellular information processing. In this work, we have investigated the RNA-binding protein YlxR. This protein is widely conserved in bacteria and strongly constitutively expressed in B. subtilis suggesting an important function. We have identified the RNA subunit of the essential RNase P as the binding partner of YlxR. The main activity of RNase P is the processing of 5′ ends of pre-tRNAs. In vitro processing assays demonstrated that the presence of YlxR results in reduced RNase P activity. Chemical cross-linking studies followed by in silico docking analysis and experiments with site-directed mutant proteins suggest that YlxR binds to the region of the RNase P RNA that is important for binding and cleavage of the pre-tRNA substrate. We conclude that the YlxR protein is a novel interaction partner of the RNA subunit of RNase P that serves to finetune RNase P activity to ensure appropriate amounts of mature tRNAs for translation. We rename the YlxR protein RnpM for RNase P modulator.

Interactors and effects of overexpressing YlxR/RpnM, a conserved RNA binding protein in cyanobacteria

“…We found two highly enriched transcripts, RNase P RNA and the 3’ segment of repA. The first result is consistent with findings for Bacillus [28] and indeed suggests a conserved function as a modulator of RNase P activity also in cyanobacteria. This interaction probably leads indirectly to the enrichment of RnpA, the protein component of RNase P, which is also bound to the RNase P RNA ( Figure 6 ).…”

“…Interestingly, a complex between a protein-only RNase P enzyme and the tRNA methyl transferases TRM1A and TRM1B has recently been described in the plant Arabidopsis Recently, an unrelated modulator of enzymatic activity has also been described for RNase E in cyanobacteria [58]. According to the work of Wicke et al [28] and based on the results of the present study, YlxR/Ssr1238 is another example of this category of regulatory proteins, but it primarily interacts with RNase P.…”

“…Initial evidence for YlxR proteins as RBPs was inferred from the structural analysis of the homolog in S. pneumoniae [23] and the in-gradient distribution of the C. difficile protein [24]. The B. subtilis YlxR protein has been reported as a nucleoid-associated protein that regulates nearly 400 genes [25][26][27], and more recently to bind to the RNA subunit of the ribonuclease P and modulate its activity [28]. Consequently, the B. subtilis YlxR protein has been renamed as RNase P modulator (RnpM) [28].…”

“…Homologs were also detected in the genomes of diatom-associated symbionts Another difference to the Bacillus protein is that residues K11, V13, T15, and D53, which are critical for RNA binding in RnpM [28], appear to have been replaced in Ssr1238/YlxR (residues R7, L9, C11 and T47, Figure 1A). However, residues corresponding to R9, R19 and the GRGA(W/Y motif of the S. pneumoniae YlxR are well conserved.…”

“…The B. subtilis YlxR protein has been reported as a nucleoid-associated protein that regulates nearly 400 genes [25][26][27], and more recently to bind to the RNA subunit of the ribonuclease P and modulate its activity [28]. Consequently, the B. subtilis YlxR protein has been renamed as RNase P modulator (RnpM) [28].…”