Phylogenetic analysis and evolution of RNase P RNA in proteobacteria

Abstract

The secondary structures of the eubacterial RNase P RNAs are being elucidated by a phylogenetic comparative approach. Sequences of genes encoding RNase P RNA from each of the recognized subgroups (a, I, y, and 8) of the proteobacteria have now been determined. These sequences allow the refinement, to nearly the base pair level, of the phylogenetic model for RNase P RNA secondary structure. Evolutionary change among the RNase P RNAs was found to occur primarily in four discrete structural domains that are peripheral to a highly conserved core structure. The new sequences were used to examine critically the proposed similarity (C. Guerrier-Takada, N. Lumelsky, and S. Altman, Science 246:1578-1584) between a portion of RNase P RNA and the "exit site" of the 23S rRNA of Escherichia cofi. Phylogenetic comparisons indicate that these sequences are not homologous and that any similarity in the structures is, at best, tenuous.RNase P cleaves leader sequences from precursor tRNAs to generate the mature 5' end of tRNA (see references 1 and 19 for reviews). In the eubacteria Bacillus subtilis and Escherichia coli, RNase P is composed of a small protein (119 amino acids) and a large RNA (-400 nucleotides). Although RNase P functions as a ribonucleoprotein in vivo, the RNA alone is an efficient and accurate catalyst at elevated concentrations of salt in vitro. RNase P contains the only RNA known to act catalytically in vivo, in the sense that each molecule of RNase P RNA acts on many substrate molecules.An accurate model for the structure of eubacterial RNase P RNA is a prerequisite for understanding the mechanism of substrate recognition, catalytic activity, and evolution of this ribozyme. A model for the secondary structure of RNase P RNA in eubacteria was originally derived by phylogenetic comparative analysis of the sequences of genes encoding RNase P RNA from several members of the y subdivision of the proteobacteria and four species of gram-positive eubacteria of the genus Bacillus (12). On the basis of the criteria used for analysis of ribosomal RNA structure (8), covariation of two base pairings within a potential continuous helix was considered proof of the occurrence of that helix. In order to increase the resolution of this model, we have cloned and sequenced the genes for RNase P RNA from a phylogenetically diverse sampling of the following additional proteobacteria: the a proteobacteria Agrobacterium tumefaciens and Rhodospirillum rubrum, the P proteobacterium Alcaligenes eutrophus, the y proteobacterium Chromatium vinosum, and the 8 proteobacterium Desulfovibrio desulfuricans (Fig. 1). Together with the previously obtained gene sequences for RNase P RNA, these analyses complete a sampling of each of the major phylogenetic branches of proteobacteria.* Corresponding author. Nucleic acid extraction. Extracts for the isolation of genomic DNAs were prepared by resuspending the cells in STE (10 mM Tris-Cl [pH 91, 100 mM NaCl, 1 mM EDTA) (15 ml/g of cells [wet weight]), treating them with lysozyme (1 mg/ml) for 5 min ...

Site-specific cleavage by metal ion cofactors and inhibitors of M1 RNA, the catalytic subunit of RNase P from Escherichia coli.

“…Consistent with this notion is the fact that sites III and V (but not IV) in M1 RNA are found (Fig. 3A) within a phylogenetically conserved structural feature (16). Furthermore, P RNA also is cleaved by metal ions in the region that corresponds to sites I and III.…”

Solution structure of RNase P RNA

“…1C). This RNA is catalytically active under high ionic strength, characteristic of all of bacterial RNase P RNA species (Brown et al 1991) and its P6 element is predicted to contain 7 bp, potentially more stable than the 4-bp helix P6 of E. coli.…”

“…DNA constructs for the expression of RNA were generated by PCR from plasmids pBstHH 2 (Kazantsev et al 2005), pDW98 , or genomic DNA of A. tumefaciens strain DSM 30150 (Brown et al 1991), cloned under T7 RNA polymerase promoter into EcoRI/KpnI digest of pRAV-23 vector (Batey and Kieft 2007) and verified by sequencing. PCR of the templates for in vitro transcription was performed as described (Batey and Kieft 2007).…”

The RNase P family

“…16,17 The type "A2" RNase P RNAs of most species of β-proteobacteria lack both P13 and P14. 12 Associated with this loss are changes in the conserved sequences at the base of P12 including the loss of the bulged AA doublet. The bases of P13 and P14 stack to create a single structural element; the loop of P14 interacts with P8, providing stabilization of the P7/P8/P9/P10 cruciform.…”

“…The secondary structure of RNase P RNA in bacteria has been wellestablished primarily on the basis of comparative analysis of several hundred sequences. [10][11][12][13] Two distinct secondary structure types predominate in bacteria: Type A and Type B. Within each type, the RNAs share a common conserved "body plan", often decorated by volatile insertions and deletions.…”