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.     

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.     

In search of RNase P RNA from microbial genomes

A simple procedure has been developed to quickly retrieve and validate the DNA sequence encoding the RNA subunit of ribonuclease P (RNase P RNA) from microbial genomes. RNase P RNA sequences were identified from 94% of bacterial and archaeal complete genomes where previously no RNase P RNA was annotated. A sequence was found in camelpox virus, highly conserved in all orthopoxviruses (including smallpox virus), which could fold into a putative RNase P RNA in terms of conserved primary features and secondary structure. New structure features of RNase P RNA that enable one to distinguish bacteria from archaea and eukarya were found. This RNA is yet another RNA that can be a molecular criterion to divide the living world into three domains (bacteria, archaea, and eukarya). The catalytic center of this RNA, and its detection from some environmental whole genome shotgun sequences, is also discussed.     

Phylogenetic-comparative analysis of the eukaryal ribonuclease P RNA

Ribonuclease P (RNase P) is the ribonucleoprotein enzyme that cleaves 5'-leader sequences from precursor-tRNAs. Bacterial and eukaryal RNase P RNAs differ fundamentally in that the former, but not the latter, are capable of catalyzing pre-tRNA maturation in vitro in the absence of proteins. An explanation of these functional differences will be assisted by a detailed comparison of bacterial and eukaryal RNase P RNA structures. However, the structures of eukaryal RNase P RNAs remain poorly characterized, compared to their bacterial and archaeal homologs. Hence, we have taken a phylogenetic-comparative approach to refine the secondary structures of eukaryal RNase P RNAs. To this end, 20 new RNase P RNA sequences have been determined from species of ascomycetous fungi representative of the genera Arxiozyma, Clavispora, Kluyveromyces, Pichia, Saccharomyces, Saccharomycopsis, Torulaspora, Wickerhamia, and Zygosaccharomyces. Phylogenetic-comparative analysis of these and other sequences refines previous eukaryal RNase P RNA secondary structure models. Patterns of sequence conservation and length variation refine the minimum-consensus model of the core eukaryal RNA structure. In comparison to bacterial RNase P RNAs, the eukaryal homologs lack RNA structural elements thought to be critical for both substrate binding and catalysis. Nonetheless, the eukaryal RNA retains the main features of the catalytic core of the bacterial RNase P. This indicates that the eukaryal RNA remains intrinsically a ribozyme.     

Phylogenetic comparative chemical footprint analysis of the interaction between ribonuclease P RNA and tRNA.

Ribonuclease P RNA is the catalytic moiety of the ribonucleoprotein enzyme that endonucleolytically cleaves precursor sequences from the 5' ends of pre-tRNAs. The bacterial RNase P RNA-tRNA complex was examined with a footprinting approach, utilizing chemical modification to determine RNase P RNA nucleotides that potentially contact tRNA. RNase P RNA was modified with dimethylsulfate or kethoxal in the presence or absence of tRNA, and sites of modification were detected by primer extension. Comparison of the results reveals RNase P bases that are protected from modification upon binding tRNA. Analyses were carried out with RNase P RNAs from three different bacteria: Escherichia coli, Chromatium vinosum and Bacillus subtilis. Discrete bases of these RNAs that lie within conserved, homologous portions of the secondary structures are similarly protected. One protection among all three RNAs was attributed to the precursor segment of pre-tRNA. Experiments using pre-tRNAs containing precursor segments of variable length demonstrate that a precursor segment of only 2-4 nucleotides is sufficient to confer this protection. Deletion of the 3'-terminal CCA sequence of tRNA correlates with loss of protection of a particular loop in the RNase P RNA secondary structure. Analysis of mutant tRNAs containing sequential 3'-terminal deletions suggests a relative orientation of the bound tRNA CCA to that loop.     

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.     

Use of RNA Secondary Structure for Studying the Evolution of RNase P and RNase MRP

Secondary structure is evaluated for determining evolutionary relationships between catalytic RNA molecules that are so distantly related they are scarcely alignable. The ribonucleoproteins RNase P (P) and RNase MRP (MRP) have been suggested to be evolutionarily related because of similarities in both function and secondary structure. However, their RNA sequences cannot be aligned with any confidence, and this leads to uncertainty in any trees inferred from sequences. We report several approaches to using secondary structures for inferring evolutionary trees and emphasize quantitative tests to demonstrate that evolutionary information can be recovered. For P and MRP, three hypotheses for the relatedness are considered. The first is that MRP is derived from P in early eukaryotes. The next is that MRP is derived from P from an early endosymbiont. The third is that both P and MRP evolved in the RNA-world (and the need for MRP has since been lost in prokaryotes). Quantitative comparisons of the pRNA and mrpRNA secondary structures have found that the possibility of an organellar origin of MRP is unlikely. In addition, comparison of secondary structures support the identity of an RNase P–like sequence in the maize chloroplast genome. Overall, it is concluded that RNA secondary structure is useful for evaluating evolutionary relatedness, even with sequences that cannot be aligned with confidence. Received: 19 July 1999 / Accepted: 3 May 2000     

Partial characterization of an RNA component that copurifies with Saccharomyces cerevisiae RNase P.

Saccharomyces cerevisiae cellular RNase P is composed of both protein and RNA components that are essential for activity. The isolated holoenzyme contains a highly structured RNA of 369 nucleotides that has extensive sequence similarities to the 286-nucleotide RNA associated with Schizosaccharomyces pombe RNase P but bears little resemblance to the analogous RNA sequences in procaryotes or S. cerevisiae mitochondria. Even so, the predicted secondary structure of S. cerevisiae RNA is strikingly similar to the bacterial phylogenetic consensus rather than to previously predicted structures of other eucaryotic RNase P RNAs.     

Comparative structure analysis of vertebrate ribonuclease P RNA.

Ribonuclease P cleaves 5'-precursor sequences from pre-tRNAs. All cellular RNase P holoenzymes contain homologous RNA elements; the eucaryal RNase P RNA, in contrast to the bacterial RNA, is catalytically inactive in the absence of the protein component(s). To understand the function of eucaryal RNase P RNA, knowledge of its structure is needed. Considerable effort has been devoted to comparative studies of the structure of this RNA from diverse organisms, including eucaryotes, primarily fungi, but also a limited set of vertebrates. The substantial differences in the sequences and structures of the vertebrate RNAs from those of other organisms have made it difficult to align the vertebrate sequences, thus limiting comparative studies. To expand our understanding of the structure of diverse RNase P RNAs, we have isolated by PCR and sequenced 13 partial RNase P RNA genes from 11 additional vertebrate taxa representing most extant major vertebrate lineages. Based on a recently proposed structure of the core elements of RNase P RNA, we aligned the sequences and propose a minimum consensus secondary structure for the vertebrate RNase P RNA.     

The RNase P RNA from cyanobacteria: short tandemly repeated repetitive (STRR) sequences are present within the RNase P RNA gene in heterocyst-forming cyanobacteria.

The RNase P RNA gene (rnpB) from 10 cyanobacteria has been characterized. These new RNAs, together with the previously available ones, provide a comprehensive data set of RNase P RNA from diverse cyanobacterial lineages. All heterocystous cyanobacteria, but none of the non-heterocystous strains analyzed, contain short tandemly repeated repetitive (STRR) sequences that increase the length of helix P12. Site-directed mutagenesis experiments indicate that the STRR sequences are not required for catalytic activity in vitro. STRR sequences seem to have recently and independently invaded the RNase P RNA genes in heterocyst-forming cyanobacteria because closely related strains contain unrelated STRR sequences. Most cyanobacteria RNase P RNAs lack the sequence GGU in the loop connecting helices P15 and P16 that has been established to interact with the 3'-end CCA in precursor tRNA substrates in other bacteria. This character is shared with plastid RNase P RNA. Helix P6 is longer than usual in most cyanobacteria as well as in plastid RNase P RNA.     

Phylogenetic analysis of the structure of RNase MRP RNA in yeasts

RNase MRP is a ribonucleoprotein enzyme involved in processing precursor rRNA in eukaryotes. To facilitate our structure-function analysis of RNase MRP from Saccharomyces cerevisiae, we have determined the likely secondary structure of the RNA component by a phylogenetic approach in which we sequenced all or part of the RNase MRP RNAs from 17 additional species of the Saccharomycetaceae family. The structure deduced from these sequences contains the helices previously suggested to be common to the RNA subunit of RNase MRP and the related RNA subunit of RNase P, an enzyme cleaving tRNA precursors. However, outside this common region, the structure of RNase MRP RNA determined here differs from a previously proposed universal structure for RNase MRPs. Chemical and enzymatic structure probing analyses were consistent with our revised secondary structure. Comparison of all known RNase MRP RNA sequences revealed three regions with highly conserved nucleotides. Two of these regions are part of a helix implicated in RNA catalysis in RNase P, suggesting that RNase MRP may cleave rRNA using a similar catalytic mechanism.     

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.     

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.     

Structural implications of novel diversity in eucaryal RNase P RNA

Previous eucaryotic RNase P RNA secondary structural models have been based on limited diversity, representing only two of the approximately 30 phylogenetic kingdoms of the domain Eucarya. To elucidate a more generally applicable structure, we used biochemical, bioinformatic, and molecular approaches to obtain RNase P RNA sequences from diverse organisms including representatives of six additional kingdoms of eucaryotes. Novel sequences were from acanthamoeba (Acathamoeba castellanii, Balamuthia mandrillaris, Filamoeba nolandi), animals (Caenorhabditis elegans, Drosophila melanogaster), alveolates (Theileria annulata, Babesia bovis), conosids (Dictyostelium discoideum, Physarum polycephalum), trichomonads (Trichomonas vaginalis), microsporidia (Encephalitozoon cuniculi), and diplomonads (Giardia intestinalis). An improved alignment of eucaryal RNase P RNA sequences was assembled and used for statistical and comparative structural analysis. The analysis identifies a conserved core structure of eucaryal RNase P RNA that has been maintained throughout evolution and indicates that covariation in size occurs between some structural elements of the RNA. Eucaryal RNase P RNA contains regions of highly variable length and structure reminiscent of expansion segments found in rRNA. The eucaryal RNA has been remodeled through evolution as a simplified version of the structure found in bacterial and archaeal RNase P RNAs.     

Structure of ribonuclease P--a universal ribozyme

Ribonuclease P (RNase P) is one of only two known universal ribozymes and was one of the first ribozymes to be discovered. It is involved in RNA processing, in particular the 5' maturation of tRNA. Unlike most other natural ribozymes, it recognizes and cleaves its substrate in trans. RNase P is a ribonucleoprotein complex containing one RNA subunit and as few as one protein subunit. It has been shown that, in bacteria and in some archaea, the RNA subunit alone can support catalysis. The structure and function of bacterial RNase P RNA have been studied extensively, but the detailed catalytic mechanism is not yet fully understood. Recently, structures of one of the structural domains and of the entire RNA component of RNase P from two different bacteria have been described. These structures provide the first atomic-level information on the structural assembly of the RNA component, and the regions involved in substrate recognition and catalysis. Comparison of these structures reveals a highly conserved core that comprises two universally conserved structural modules. Interestingly, the same structural core can be found in the context of different scaffolds.     

Secondary structure probing of the human RNase MRP RNA reveals the potential for MRP RNA subsets

RNase MRP is a ribonucleoprotein endoribonuclease involved in eukaryotic pre-rRNA processing. The enzyme possesses an RNA subunit, structurally related to that of RNase P RNA, that is thought to be catalytic. RNase MRP RNA sequences from Saccharomycetaceae species are structurally well defined through detailed phylogenetic and structural analysis. In contrast, higher eukaryote MRP RNA structure models are based on comparative sequence analysis of only five sequences and limited probing data. Detailed structural analysis of the Homo sapiens MRP RNA, entailing enzymatic and chemical probing, is reported. The data are consistent with the phylogenetic secondary structure model and demonstrate unequivocally that higher eukaryote MRP RNA structure differs significantly from that reported for Saccharomycetaceae species. Neither model can account for all of the known MRP RNAs and we thus propose the evolution of at least two subsets of RNase MRP secondary structure, differing predominantly in the predicted specificity 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.     

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.     

A microdiversity study of anammox bacteria reveals a novel Candidatus Scalindua phylotype in marine oxygen minimum zones

The anaerobic oxidation of ammonium (anammox) contributes significantly to the global loss of fixed nitrogen and is carried out by a deep branching monophyletic group of bacteria within the phylum Planctomycetes. Various studies have implicated anammox to be the most important process responsible for the nitrogen loss in the marine oxygen minimum zones (OMZs) with a low diversity of marine anammox bacteria. This comprehensive study investigated the anammox bacteria in the suboxic zone of the Black Sea and in three major OMZs (off Namibia, Peru and in the Arabian Sea). The diversity and population composition of anammox bacteria were investigated by both, the 16S rRNA gene sequences and the 16S-23S rRNA internal transcribed spacer (ITS). Our results showed that the anammox bacterial sequences of the investigated samples were all closely related to the Candidatus Scalindua genus. However, a greater microdiversity of marine anammox bacteria than previously assumed was observed. Both phylogenetic markers supported the classification of all sequences in two distinct anammox bacterial phylotypes: Candidatus Scalindua clades 1 and 2. Scalindua 1 could be further divided into four distinct clusters, all comprised of sequences from either the Namibian or the Peruvian OMZ. Scalindua 2 consisted of sequences from the Arabian Sea and the Peruvian OMZ and included one previously published 16S rRNA gene sequence from Lake Tanganyika and one from South China Sea sediment (97.9-99.4% sequence identity). This cluster showed only 相似文献