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

Abstract

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 post-transcriptionally 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.

Suppression of theEscherichia coli rnpA49conditionally lethal phenotype by different compensatory mutations

“…As the rnpA49 mutation is hypothesized to reduce the efficiency with which C5 A49 assembles with the M1 RNA, overexpression of either subunit of the mutant RNase P heterodimer likely shifts the equilibrium of assembly towards formation of the RNase P holoenzyme. This is also consistent with previous work demonstrating that wild-type rnpA overexpression increases the steady-state levels of the RNase P holoenzyme Gößringer et al 2023).…”

Multiple structural flavors of RNase P in precursor tRNA processing

“…Some prokaryotes also harbor protein‐only variants of RNase P, collectively referred to as Homologs of Aquifex RNase P (HARP; discussed in Section 4), owing to their first discovery in Aquifex aeolicus (Nickel et al, 2017). HARPs are remarkably smaller than eukaryotic PRORPs and have either replaced the classical RNP RNase P as in Aquifex aeolicus or co‐exist with RNP RNase P as in many archaea (Lechner et al, 2014; Li & Altman, 2004a; Marszalkowski et al, 2008; Nickel et al, 2017; Schwarz et al, 2019; Swanson, 2001; Willkomm et al, 2002) and some bacteria (Gossringer et al, 2023), wherein the major RNase P activity still resides with the RNP RNase P (Gossringer et al, 2023; Schwarz et al, 2019). Thus, multiple variants of large and small forms of RNase P exist in all forms of life found in nature.…”