An unusual mechanism of bacterial gene expression revealed for the RNase P protein of Thermus strains

Abstract

The RNase P protein gene (rnpA) completely overlaps the rpmH gene (encoding ribosomal protein L34) out of frame in the thermophilic bacterium Thermus thermophilus. This results in the synthesis of an extended RNase P protein (C5) of 163 aa and, by inference, of 240 aa in the related strain Thermus filiformis. Start codons of rnpA and rpmH, apparently governed by the same ribosome binding site, are separated by only 4 nt, which suggests a regulatory linkage between L34 and C5 translation and, accordingly, between ribosome and RNase P biosynthesis. Within the sequence encoding the N-terminal extensions and downstream of rpmH, several Thermus species exhibit in-frame deletions͞inser-tions, suggesting relaxed constraints for sequence conservation in this region. Roughly the N-terminal third of T. thermophilus C5 was further shown to be dispensable for RNase P function in vitro by using a precursor tRNA Gly substrate from the same organism. Taken together, these data reveal a mode of gene expression that is to our knowledge unprecedented in bacteria.T he ubiquitous enzyme RNase P catalyzes endonucleolytic 5Ј maturation of tRNA primary transcripts in all three domains of life (Archaea, Bacteria, and Eukarya) and in mitochondria and chloroplasts (1, 2). Bacterial RNase P enzymes are composed of a catalytic RNA subunit, Ϸ400 nt in length, and a single small protein of typically 120 aa (3). The RNA subunits alone are catalytically active in vitro but require elevated salt concentrations to compensate for the absence of the protein subunit (4). RNase P holoenzymes are highly efficient catalysts, consistent with their generally low cellular abundance (2).In the majority of bacteria, the rnpA gene encoding the RNase P protein has been identified immediately downstream of the gene for the ribosomal protein L34 (rpmH) and close to the origin of replication oriC (5-7). In Escherichia coli, rpmH and rnpA were shown to be part of the same operon, with two major promoters preceding the rpmH structural gene (7-9). E. coli L34 is produced in excess over the RNase P protein (termed C5), which appears to be regulated at the transcriptional and the translational level. First, three mRNA species derived from the rpmH-rnpA operon were detected, two shorter ones lacking the rnpA cistron and a longer and much less abundant one including it (9). Second, the rnpA codon usage does not correspond to that of highly expressed E. coli genes, such as rpmH (7).We have investigated rnpA expression in thermophilic bacteria of the genus Thermus. This work led to the discovery of an unprecedented mode of (nonviral) gene expression in bacteria: rnpA completely overlaps rpmH in a different reading frame, resulting in the synthesis of unusually long RNase P proteins (163 aa in T. thermophilus). Materials and MethodsFor a detailed description of bacterial strains, cloning procedures, plasmid constructs, and protein and RNA preparation, see Supporting Text, which is published as supporting information on the PNAS web site, www.pnas.org. Transformation of ...

Overlapping Genes Produce Proteins with Unusual Sequence Properties and Offer Insight into De Novo Protein Creation

“…We hope to obtain further insights from other organisms. (22). Earlier observations on the properties of proteins encoded by overlapping genes.…”

Function of heterologous and truncated RNase P proteins in Bacillus subtilis

“…Additionally, the XL version of the D. vulgaris P protein (Fig. 1A) served as a prototype for N‐terminal sequence extensions, naturally found in bacteria of the genus Thermus (Feltens et al ., 2003). Because all three variants were functional in B. subtilis (Table 1), we conclude that these sequence extensions and insertions do not interfere with holoenzyme assembly or substrate binding.…”

“…The N‐terminus, although close to the RNA surface in the model, is flexible (Spitzfaden et al ., 2000; Kazantsev et al ., 2003). This flexibility offers an explanation whysubstantial N‐terminal extensions as in Dvul‐XL or in T. thermophilus RnpA (Feltens et al ., 2003) can be accommodated.…”

Engineering gene overlaps to sustain genetic constructs in vivo

“…Present in all domains of life, from phages [4] and bacteria [14,12] to vertebrates [24] including mammals [39], overlapping genes present a fascinating puzzle. They were discovered in the first organism ever sequenced, the phage ΦX174 [4,40], but the selective pressures and mechanisms leading to their evolution remained elusive for a long time.…”