Thermostable RNase P RNAs lacking P18 identified in the Aquificales

Abstract

The RNase P RNA (rnpB) and protein (rnpA) genes were identified in the two Aquificales Sulfurihydrogenibium azorense and Persephonella marina. In contrast, neither of the two genes has been found in the sequenced genome of their close relative, Aquifex aeolicus. As in most bacteria, the rnpA genes of S. azorense and P. marina are preceded by the rpmH gene coding for ribosomal protein L34. This genetic region, including several genes up-and downstream of rpmH, is uniquely conserved among all three Aquificales strains, except that rnpA is missing in A. aeolicus. The RNase P RNAs (P RNAs) of S. azorense and P. marina are active catalysts that can be activated by heterologous bacterial P proteins at low salt. Although the two P RNAs lack helix P18 and thus one of the three major interdomain tertiary contacts, they are more thermostable than Escherichia coli P RNA and require higher temperatures for proper folding. Related to their thermostability, both RNAs include a subset of structural idiosyncrasies in their S domains, which were recently demonstrated to determine the folding properties of the thermostable S domain of Thermus thermophilus P RNA. Unlike 16S rRNA phylogeny that has placed the Aquificales as the deepest lineage of the bacterial phylogenetic tree, RNase P RNA-based phylogeny groups S. azorense and P. marina with the green sulfur, cyanobacterial, and d/e proteobacterial branches.

Archaeal‐Bacterial Chimeric RNase P RNAs: Towards Understanding RNA's Architecture, Function and Evolution

“…The C domain of M. thermautotrophicus P RNA, as for all archaeal and also some bacterial P RNAs,31, 32 lacks the P18 element that enables formation of the L18–P8 interdomain contact. We reasoned that this interaction could further stabilize interdomain orientation in the chimeric P RNA, in addition to the L9–P1 strut, thus improving catalytic performance.…”

5′-End maturation of tRNA in Aquifex aeolicus

“…The rnpA gene is absent from the genome of A. aeolicus, but is present in Sulfurihydrogenibium azorense and Persephonella marina, the only two other Aquificales with fully assembled genome sequences (Marszalkowski et al, 2006). Genome sequences are not available for H. thermophilus, A. pyrophilus and T. ruber.…”

“…However, a major deviation between A. aeolicus and the majority of bacteria concerns the identity of the nucleotide upstream of the 'RNase P' cleavage site: whereas a pyrimidine, preferentially U, at nt -1 (or at -2 in the case of tRNA His and tRNA SeCys ) prevails in most bacteria (Brä nnvall et al, 2003;Zahler et al, 2003), 86.4% (38 out of 44 tRNAs) of the tRNA genes in A. aeolicus encode an A residue at this position. However, in the Aquificales S. azorense and P. marina, where we identified bacterial RNase P RNA and protein genes (Marszalkowski et al, 2006), the bias in favor of an A residue upstream of the RNase P cleavage site is abandoned, as these tRNA genes follow the bacterial mainstream of the 41 tRNAs identified in S. azorense, 26 (63.4%) encode a U, 7 a C and only 8 an A residue at this position; likewise, the distribution in the 40 identified tRNA genes in P. marina is 27=U (67.5%), 7=A, 3=C and 3=G (G. Steger and R.K. Hartmann, unpublished results). The bias for adenosine upstream of the cleavage site in A. aeolicus ptRNAs points to mechanistic differences in substrate recognition relative to most bacteria, which prompted us to inspect the situation in other Aquificales.…”

Structural basis of a ribozyme's thermostability: P1–L9 interdomain interaction in RNase P RNA

“…Activity of RNase P holoenzymes was measured in buffer KN (20 mM HEPES, 150 mM NH 4 OAc, 2 mM spermidine, 0.05 mM spermine, 4 mM b-mercaptoethanol at pH 7.4 at 37°C) containing 2 or 4.5 mM Mg(OAc) 2 (buffer KN2 and KN4.5; Wegscheid and Hartmann 2006), at concentrations of 10 nM P RNA, 100 nM substrate, and 40 nM P protein (recombinantly expressed and purified exactly as in Marszalkowski et al 2006). Prior to the reaction, substrate and P RNA were preincubated in the reaction buffer (substrate as above, P RNA for 5 min at 55°C and 50 min at 37°C); P protein was then added to the P RNA and preincubation continued for another 5 min at 37°C, after which substrate was added.…”

“…We recently reported that P RNAs from thermophilic bacteria share a 59-GYAA L9 tetraloop and a P1 receptor site consisting of a G-C base-pair (bp) tandem, a combination not present in other bacteria (Marszalkowski et al 2006). Also, helices P1 and P9 are stabilized in P RNAs from thermophiles by helix extension and/or deletion of nucleotide bulges.…”