New insight into RNase P RNA structure from comparative analysis of the archaeal RNA.

A detailed comparative analysis of archaeal RNase P RNA structure and a comparison of the resulting structural information with that of the bacterial RNA reveals that the archaeal RNase P RNAs are strikingly similar to those of Bacteria. The differences between the secondary structure models of archaeal and bacterial RNase P RNA have largely disappeared, and even variation in the sequence and structure of the RNAs are similar in extent and type. The structure of the cruciform (P7-11) has been reevaluated on the basis of a total of 321 bacterial and archaeal sequences, leading to a model for the structure of this region of the RNA that includes an extension to P11 that consistently organizes the cruciform and adjacent highly-conserved sequences.

RNase P RNAs from some Archaea are catalytically active.

The RNA subunits of RNase Ps of Archaea and eukaryotes have been thought to depend fundamentally on protein for activity, unlike those of Bacteria that are capable of efficient catalysis in the absence of protein. Although the eukaryotic RNase P RNAs are quite different than those of Bacteria in both sequence and structure, the archaeal RNAs generally contain the sequences and structures of the bacterial, phylogenetically conserved catalytic core. A spectrum of archaeal RNase P RNAs were therefore tested for activity in a wide range of conditions. Many remain inactive in ionically extreme conditions, but catalytic activity could be detected from those of the methanobacteria, thermococci, and halobacteria. Chimeric holoenzymes, reconstituted from the Methanobacterium RNase P RNA and the Bacillus subtilis RNase P protein subunits, were functional at low ionic strength. The properties of the archaeal RNase P RNAs (high ionic-strength requirement, low affinity for substrate, and catalytic reconstitution by bacterial RNase P protein) are similar to synthetic RNase P RNAs that contain all of the catalytic core of the bacterial RNA but lack phylogenetically variable, stabilizing elements.

Evolutionary variation in bacterial RNase P RNAs.

Sequences encoding RNase P RNAs from representatives of the last remaining classical phyla of Bacteria have been determined, completing a general phylogenetic survey of RNase P RNA sequence and structure. This broad sampling of RNase P RNAs allows some refinement of the secondary structure, and reveals patterns in the evolutionary variation of sequences and secondary structures. Although the sequences range from 100 to <25% identical to one another, and although only 40 of the nucleotides are invariant, there is considerable conservation of the underlying core of the RNA sequence. RNase P RNAs, like group I intron RNAs but unlike ribosomal RNAs, transfer RNAs or other highly conserved RNAs, are quite variable in secondary structure outside of this conserved structural core. Conservative regions of the RNA evolve by substitution of apparently interchangeable alternative structures, rather than the insertion and deletion of helical elements that occurs in the more variable regions of the RNA. In a remarkable case of convergent molecular evolution, most of the unusual structural elements of type B RNase P RNAs of the low G+C Gram-positive Bacteria have evolved independently in Thermomicrobium roseum , a member of the green non-sulfur Bacteria.

Intracellular localization and unique conserved sequences of three small nucleolar RNAs.

Three human small nucleolar RNAs (snoRNAs), E1, E2 and E3, were reported earlier that have unique sequences, interact directly with unique segments of pre-rRNA in vivo and are encoded in introns of protein genes. In the present report, human and frog E1, E2 and E3 RNAs injected into the cytoplasm of frog oocytes migrated to the nucleus and specifically to the nucleolus. This indicates that the nucleolar and nuclear localization signals of these snoRNAs reside within their evolutionarily conserved segments. Homologs of these snoRNAs from several vertebrates were sequenced and this information was used to develop RNA secondary structure models. These snoRNAs have unique phylogenetically conserved sequences.

Structure and evolution of ribonuclease P RNA in Gram-positive bacteria.

The sequences and structures of RNase P RNAs of some Gram-positive bacteria, e.g. Bacillus subtilis, are very different than those of other bacteria. In order to expand our understanding of the structure and evolution of RNase P RNA in Gram-positive bacteria, gene sequences encoding RNase P RNAs from 10 additional species from this evolutionary group have been determined, doubling the number of sequences available for comparative analysis. The enlarged data set allows refinement of the secondary structure model of these unusual RNase P RNAs and the identification of potential tertiary interactions between P10.1 and L12, and between L5.1 and L15.1. The newly-obtained sequences suggest that RNase P RNA underwent an abrupt, dramatic restructuring in the ancestry of the low-G+C Gram-positive bacteria after the divergence of the branches leading to the 'Clostridia and relatives' and the remaining low-G+C Gram-positive species. The unusual structures of the RNase P RNAs of Mycoplasma hyopneumoniae and M.floccularre are apparently derived from RNAs with Bacillus-like structure rather than from intermediate, partially restructured ancestral RNAs. The structure of the RNase P RNA from the photosynthetic Heliobacillus mobilis supports the relationship of this specie with Bacillus and Staphylococcus rather than the 'Clostridia and relatives' as suggested by the sequences of their small-subunit ribosomal RNAs.

Mycoplasma fermentans simplifies our view of the catalytic core of ribonuclease P RNA.

The catalytic RNA moiety of (eu)bacterial RNase P is responsible for cleavage of the 5' leader sequence from precursor tRNAs. We report the sequence, the catalytic properties, and a phylogenetic-comparative structural analysis of the RNase P RNA from Mycoplasma fermentans, at 276 nt the smallest known RNase P RNA. This RNA is noteworthy in that it lacks a stem-loop structure (helix P12) that was thought previously to be universally present in bacterial RNase P RNAs. This finding suggests that helix P12 is not required for catalytic activity in vivo. In order to test this possibility in vitro, the kinetic properties of M. fermentans RNase P RNA and a mutant Escherichia coli RNase P RNA that was engineered to lack helix P12 were determined. These RNase P RNAs are catalytically active with efficiencies (Kcat/Km) comparable to that of native E. coli RNase P RNA. These results show that helix P12 is dispensable in vivo in some organisms, and therefore is unlikely to be essential for the mechanism of RNase P action. The notion that all phylogenetically volatile structures in RNase P RNA are dispensable for the catalytic mechanism was tested. A synthetic RNA representing the phylogenetic minimum RNase P RNA was constructed by deleting all evolutionarily variable structures from the M. fermentans RNA. This simplified RNA (Micro P RNA) was catalytically active in vitro with approximately 600-fold decrease in catalytic efficiency relative to the native RNA.

Comparative analysis of ribonuclease P RNA structure in Archaea.

Although the structure of the catalytic RNA component of ribonuclease P has been well characterized in Bacteria, it has been little studied in other organisms, such as the Archaea. We have determined the sequences encoding RNase P RNA in eight euryarchaeal species: Halococcus morrhuae, Natronobacterium gregoryi, Halobacterium cutirubrum, Halobacteriurn trapanicum, Methanobacterium thermoautotrophicum strains deltaH and Marburg, Methanothermus fervidus and Thermococcus celer strain AL-1. On the basis of these and previously available sequences from Sulfolobus acidocaldarius, Haloferax volcanii and Methanosarcina barkeri the secondary structure of RNase P RNA in Archaea has been analyzed by phylogenetic comparative analysis. The archaeal RNAs are similar in both primary and secondary structure to bacterial RNase P RNAs, but unlike their bacterial counterparts these archaeal RNase P RNAs are not by themselves catalytically proficient in vitro.

Comparative analysis of ribonuclease P RNA using gene sequences from natural microbial populations reveals tertiary structural elements.

PCR amplification of template DNAs extracted from mixed, naturally occurring microbial populations, using oligonucleotide primers complementary to highly conserved sequences, was used to obtain a large collection of diverse RNase P RNA-encoding genes. An alignment of these sequences was used in a comparative analysis of RNase P RNA secondary and tertiary structure. The new sequences confirm the secondary structure model based on sequences from cultivated organisms (with minor alterations in helices P12 and P18), providing additional support for nearly every base pair. Analysis of sequence covariation using the entire RNase P RNA data set reveals elements of tertiary structure in the RNA; the third nucleotides (underlined) of the GNRA tetraloops L14 and L18 are seen to interact with adjacent Watson-Crick base pairs in helix P8, forming A:G/C or G:A/U base triples. These experiments demonstrate one way in which the enormous diversity of natural microbial populations can be used to elucidate molecular structure through comparative analysis.

Ribonuclease P structure and function in Archaea.

An important approach to understanding RNA-based catalytic function by ribonuclease P is the investigation of its evolutionary diversity in structure and function. Because RNase P enzymes from all organisms are thought to share common ancestry, the fundamental features of structure and biochemistry should be conserved in all of its modern forms. In contrast to the bacterial enzyme, the RNase P enzymes from Eucarya, organelles, and Archaea are poorly understood. This review describes our nascent understanding of the structure and function of RNase P in Archaea, and how this enzyme compares to its homologs in the other evolutionary Domains.

The Ribonuclease P database.

The Ribonuclease P Sequence database is a compilation of RNase P sequences, sequence alignments, secondary structures, three-dimensional models, and accessory information. In its initial form, the database contains information on RNase P RNA in bacteria and archaea, and RNase P protein in bacteria. The sequences themselves are presented phylogenetically ordered and aligned. The database also contains secondary structures of bacterial and archaeal RNAs, including specially annotated 'reference' secondary structures of Escherichia coli and Bacillus subtilis RNase P RNAs, a minimum phylogenetic consensus structure, and coordinates for models of three-dimensional structure.

Further perspective on the catalytic core and secondary structure of ribonuclease P RNA.

Phylogenetic comparative analyses of RNase P RNA-encoding gene sequences from Chlorobium limicola, Chlorobium tepidum, Bacteroides thetaiotaomicron, and Flavobacterium yabuuchiae refine the secondary structure model of the general (eu)bacterial RNase P RNA and show that a highly conserved feature of that RNA is not essential. Two helices, comprised of 2 base pairs each, are added to the secondary structure model and form part of a cruciform in the RNA. Novel sequence variations in the B. thetaiotaomicron and F. yabuuchiae RNA indicate the likelihood that all secondary structure resulting from canonical base-pairing has been detected: there are no remaining unpaired, contiguous, canonical complementarities in the structure model common to all bacterial RNase P RNAs. A nomenclature for the elements of the completed secondary structure model is proposed. The Chlorobium RNase P RNAs lack a stem-loop structure that is otherwise universally present and highly conserved in structure in other (eu)bacterial RNase P RNAs. The Chlorobium RNAs are nevertheless catalytic, with kinetic properties similar to those of RNase P RNAs of Escherichia coli and other Bacteria. Removal of this stem-loop structure from the E. coli RNA affects neither its affinity for nor its catalytic rate for cleavage of a precursor transfer RNA substrate. These results show that this structural element does not play a direct role in substrate binding or catalysis.

Characterization of the RNase P RNA of Sulfolobus acidocaldarius.

RNase P is the ribonucleoprotein enzyme that cleaves precursor sequences from the 5' ends of pre-tRNAs. In Bacteria, the RNA subunit is the catalytic moiety. Eucaryal and archaeal RNase P activities copurify with RNAs, which have not been shown to be catalytic. We report here the analysis of the RNase P RNA from the thermoacidophilic archaeon Sulfolobus acidocaldarius. The holoenzyme was highly purified, and extracted RNA was used to identify the RNase P RNA gene. The nucleotide sequence of the gene was determined, and a secondary structure is proposed. The RNA was not observed to be catalytic by itself, but it nevertheless is similar in sequence and structure to bacterial RNase P RNA. The marked similarity of the RNase P RNA from S. acidocaldarius and that from Haloferax volcanii, the other known archael RNase P RNA, supports the coherence of Archaea as a phylogenetic domain.

Characterization of ribonuclease P RNAs from thermophilic bacteria.

The catalytic RNA component of bacterial RNase P is responsible for the removal of 5' leader sequences from precursor tRNAs. As part of an on-going phylogenetic comparative characterization of bacterial RNase P, the genes encoding RNase P RNA from the thermophiles Thermotoga maritima, Thermotoga neapolitana, Thermus aquaticus, and a mesophilic relative of the latter, Deinococcus radiodurans, have been cloned and sequenced. RNAs transcribed from these genes in vitro are catalytically active in the absence of other components. Active holoenzymes have been reconstituted from the T.aquaticus and T.maritima RNAs and the protein component of RNase P from Escherichia coli. The RNase P RNAs of T.aquaticus and T.martima, synthesized in vitro, were characterized biochemically and shown to be inherently resistant to thermal disruption. Several features of these RNAs suggest mechanisms contributing to thermostability. The new sequences provide correlations that refine the secondary structure model of bacterial RNase P RNA.

Influence of metal ions on the ribonuclease P reaction. Distinguishing substrate binding from catalysis.

A high yield, photoactivated cross-linking reaction between a modified tRNA and RNase P RNA was used as a quantitative assay of substrate binding affinity. The cross-linking assay allows the effects of metal ions on substrate binding to be measured independently and in the absence of the pre-tRNA cleavage reaction. The results of this assay, in conjunction with the conventional cleavage assay, support the following conclusions about the nature of the RNase P RNA-tRNA binding interaction. (i) Monovalent cations act primarily to enhance enzyme-substrate binding, presumably by functioning as counterions. This enhancement can be attributed to a reduction in the tRNA off-rate. (ii) Although divalent cation is required for cleavage, the enzyme-substrate complex can form in the absence of divalent cation; the essential role of divalent cation in the reaction is thus catalytic. (iii) Ca2+ is as efficient as Mg2+ in promoting binding but supports catalysis only at a low rate.

Long-range structure in ribonuclease P RNA.

Phylogenetic-comparative and mutational analyses were used to elucidate the structure of the catalytically active RNA component of eubacterial ribonuclease P (RNase P). In addition to the refinement and extension of known structural elements, the analyses revealed a long-range interaction that results in a second pseudoknot in the RNA. This feature strongly constrains the three-dimensional structure of RNase P RNA near the active site. Some RNase P RNAs lack this structure but contain a unique, possibly compensating, structural domain. This suggests that different RNA structures located at different positions in the sequence may have equivalent architectural functions in RNase P RNA.

Phylogenetic analysis and evolution of RNase P RNA in proteobacteria.

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 (alpha, beta, gamma, and delta) 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, 1989) between a portion of RNase P RNA and the "exit site" of the 23S rRNA of Escherichia coli. Phylogenetic comparisons indicate that these sequences are not homologous and that any similarity in the structures is, at best, tenuous.

The RNA component of RNase P from the archaebacterium Haloferax volcanii.

RNase P, an endoribonuclease responsible for generating the mature 5' termini of tRNA precursors, is composed of both RNA and protein. It has been demonstrated that the eubacterial RNase P RNA will, under the appropriate reaction conditions, exhibit catalytic activity in vitro. Evidence has not been obtained for catalytic activity by the RNAs of eukaryotic RNase P enzymes. Using a cDNA probe prepared from RNA copurifying with RNase P activity from the archaebacterium Haloferax volcanii, we have characterized the gene encoding the RNase P RNA. The proposed transcript from this gene can assume a structure resembling the eubacterial RNase P RNA and includes many of the highly conserved sequences of these RNAs. This RNA was incapable of cleaving pre-tRNA substrates in the absence of protein under a variety of in vitro conditions. Catalytic activity was observed when this RNA was combined with the protein subunit of the Bacillus subtilis RNase P complex. Similarities among the archaebacterial, eubacterial, and eukaryotic RNase P RNA sequences and structures are discussed.

Genes encoding the 7S RNA and tRNA(Ser) are linked to one of the two rRNA operons in the genome of the extremely thermophilic archaebacterium Methanothermus fervidus.

Analysis of gene structure in the extremely thermophilic archaebacterium, Methanothermus fervidus, has revealed the presence of a cluster of stable RNA-encoding genes arranged 5'-7S RNA-tRNA(Ser)-16S rRNA-tRNA(Ala)-23S rRNA-5S rRNA. The genome of M. fervidus contains two rRNA operons but only one operon has the closely linked 7S RNA-encoding gene. The sequences upstream from the two rRNA operons are identical for 206 bp but diverge at the 3' base of the tRNA(Ser) gene. The secondary structures predicted for the M. fervidus 7S, 16S rRNA, tRNA(Ala) and tRNA(Ser) have been compared with those of functionally homologous molecules from moderately thermophilic and mesophilic archaebacteria. A consensus secondary structure for archaebacterial 7S RNAs has been developed which incorporates bases and structural features also conserved in eukaryotic signal-recognition-particle RNAs and eubacterial 4.5S RNAs.

Genes encoding 5S rRNA and tRNAs in the extremely thermophilic archaebacterium Methanothermus fervidus.

Methanothermus fervidus was shown to have two 5S rRNA-encoding genes linked in rRNA operons to 16S and 23S rRNA-encoding genes. Sequencing of a cloned 5S rRNA gene confirmed that M. fervidus is a member of the Methanobacteriales, although its 5S rRNA is also similar in both primary sequence and predicted secondary structure to the 5S rRNA of the non-methanogenic, but also extremely thermophilic archaebacterium, Thermococcus celer. Two clusters of tRNA genes have also been cloned and sequenced form M. fervidus. The smaller cluster, cloned in pET5401, is composed of 5'-tRNA(UGUThr)-tRNA(UGGPro)-tRNA(GUCAsp)-tRNA(UUUL ys)-3' and the larger cluster, cloned in pET5475, is composed of 5'-tRNA(GUUAsn)-tRNA(CAUMet)-tRNA(UUCGlu)-tRNA(UAGL eu)-tRNA(GUGHis)-3'. The encoded tRNAs, with the exception of the tRNA(Leu), translate abundant codons in M. fervidus. The tRNA genes do not contain introns or encode 3'-terminal CCA residues. Homologous clusters of tRNA genes have been sequenced from Methanococcus vannielii and Methanococcus voltae, so that comparisons of transcription signals, gene organizations and primary sequences can be made and features possibly related to thermostability identified. During evolution, a 5S rRNA gene appears to have been incorporated into the cluster of tRNA genes in the methanococci but not in M. fervidus.