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N R Pace

Publications and source records attributed to N R Pace.

At least 19 recordsLinked to original sources
PubMed1997

A molecular view of microbial diversity and the biosphere.

Over three decades of molecular-phylogenetic studies, researchers have compiled an increasingly robust map of evolutionary diversification showing that the main diversity of life is microbial, distributed among three primary relatedness groups or domains: Archaea, Bacteria, and Eucarya. The general properties of representatives of the three domains indicate that the earliest life was based on inorganic nutrition and that photosynthesis and use of organic compounds for carbon and energy metabolism came comparatively later. The application of molecular-phylogenetic methods to study natural microbial ecosystems without the traditional requirement for cultivation has resulted in the discovery of many unexpected evolutionary lineages; members of some of these lineages are only distantly related to known organisms but are sufficiently abundant that they are likely to have impact on the chemistry of the biosphere.

Archaea↗
PubMed1996

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.

Bacillus subtilis↗
PubMed1996

Perspectives on archaeal diversity, thermophily and monophyly from environmental rRNA sequences.

Phylogenetic analysis of ribosomal RNA sequences obtained from uncultivated organisms of a hot spring in Yellowstone National Park reveals several novel groups of Archaea, many of which diverged from the crenarchaeal line of descent prior to previously characterized members of that kingdom. Universal phylogenetic trees constructed with the addition of these sequences indicate monophyly of Archaea, with modest bootstrap support. The data also show a specific relationship between low-temperature marine Archaea and some hot spring Archaea. Two of the environmental sequences are enigmatic: depending upon the data set and analytical method used, these sequences branch deeply within the Crenarchaeota, below the bifurcation between Crenarchaeota and Euryarchaeota, or even as the sister group to Eukaryotes. If additional data confirm either of the latter two placements, then the organisms represented by these ribosomal RNA sequences would merit recognition as a new kingdom, provisionally named "Korarchaeota."

Archaea↗
PubMed1996

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.

Bacteria↗
PubMed1996

Mineralization of 2,4,6-trinitrophenol (picric acid): characterization and phylogenetic identification of microbial strains.

Four bacterial strains that use picric acid as their sole carbon and energy source were isolated. Mineralization of 14C-UL-picric acid showed that up to 65% of the radioactivity was released as 14CO2. HPLC and UV/Vis spectral analyses indicated complete degradation of picric acid by these organisms. HPLC and LC/MS analyses showed transient formation of 2,4-dinitrophenol during picric acid degradation. Degradation of picric acid was concomitant with stoichiometric release of three moles of nitrite per mole of picric acid. The four picric acid degraders were identified as close relatives of Nocardioides simplex (ATCC 6946) based on their small subunit (16S) rRNA gene sequences.

Base Sequence↗
PubMed1996

Identifying microbial diversity in the natural environment: a molecular phylogenetic approach.

Our knowledge of microbial biodiversity has been severely limited by relying on microorganisms that have been cultured; these represent only a tiny fraction of the microbial diversity in the environment. Recently, however, recombinant DNA and molecular phylogenetic techniques have provided methods for characterizing natural microbial communities without the need to cultivate organisms. These techniques have allowed a glimpse of the complexity of microbial communities and the huge, largely untapped, biotechnological resource that they represent.

Biotechnology↗
PubMed1996

Phylogenetic analysis of Metabacterium polyspora: clues to the evolutionary origin of daughter cell production in Epulopiscium species, the largest bacteria.

It is rare that there are molecular clues to the evolutionary origin of developmental traits. We have encountered an evolutionary juxtaposition that may explain the origin of the unique replicative morphology of Epulopiscium spp., the largest known bacteria, which reproduce by the internal production of multiple live offspring. We report here a 16S rRNA-based phylogenetic analysis of Metabacterium polyspora, a multiple-endospore-forming, uncultivated inhabitant of guinea pig cecum. Cells of M. polyspora were harvested from cecum contents by sedimentation in a Ficoll gradient and lysed. The bacterial 16S rRNA genes of this lysate were amplified by PCR. Sequence analysis of the cloned PCR products revealed two dominant, closely related 16S rRNA types. In situ hybridization of cecum contents with fluorescently labeled oligonucleotides, diagnostic of these two sequences, showed that they represent distinct strains of M. polyspora. Phylogenetic analyses of the sequences showed that M. polyspora is closely related to Epulopiscium spp. On the basis of this result and other correlations, we propose that the process of sporulation was modified in a predecessor of Epulopiscium spp. to produce live offspring instead of quiescent endospores.

Animals↗
PubMed1996

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.

Base Sequence↗
PubMed1996

In vitro selection of RNase P RNA reveals optimized catalytic activity in a highly conserved structural domain.

In vitro selection techniques are useful means of dissecting the functions of both natural and artificial ribozymes. Using a self-cleaving conjugate containing the Escherichia coli ribonuclease P RNA and its substrate, pre-tRNA (Frank DN, Harris ME, Pace NR, 1994, Biochemistry 33:10800-10808), we have devised a method to select for catalytically active variants of the RNase P ribozyme. A selection experiment was performed to probe the structural and sequence constraints that operate on a highly conserved region of RNase P: the J3/4-P4-J2/4 region, which lies within the core of RNase P and is thought to bind catalytically essential magnesium ions (Harris ME et al., 1994, EMBO J 13:3953-3963; Hardt WD et al., 1995, EMBO J 14:2935-2944; Harris ME, Pace NR, 1995, RNA 1:210-218). We sought to determine which, if any, of the nearly invariant nucleotides within J3/4-P4-J2/4 are required for ribozyme-mediated catalysis. Twenty-two residues in the J3/4-P4-J2/4 component of RNase P RNA were randomized and, surprisingly, after only 10 generations, each of the randomized positions returned to the wild-type sequence. This indicates that every position in J3/4-P4-J2/4 contributes to optimal catalytic activity. These results contrast sharply with selections involving other large ribozymes, which evolve improved catalytic function readily in vitro (Chapman KB, Szostak JW, 1994, Curr Opin Struct Biol 4:618-622; Joyce GF, 1994, Curr Opin Struct Biol 4:331-336; Kumar PKR, Ellington AE, 1995, FASEB J 9:1183-1195). The phylogenetic conservation of J3/4-P4-J2/4, coupled with the results reported here, suggests that the contribution of this structure to RNA-mediated catalysis was optimized very early in evolution, before the last common ancestor of all life.

Base Sequence↗
PubMed1995

Identification of phosphates involved in catalysis by the ribozyme RNase P RNA.

The RNA subunit of ribonuclease P (RNase P RNA) is a catalytic RNA that cleaves precursor tRNAs to generate mature tRNA 5' ends. Little is known concerning the identity and arrangement of functional groups that constitute the active site of this ribozyme. We have used an RNase P RNA-substrate conjugate that undergoes rapid, accurate, and efficient self-cleavage in vitro to probe, by phosphorothioate modification-interference, functional groups required for catalysis. We identify four phosphate oxygens where substitution by sulfur significantly reduces the catalytic rate (50-200-fold). Interference at one site was partially rescued in the presence of manganese, suggesting a direct involvement in binding divalent metal ion cofactors required for catalysis. All sites are located in conserved sequence and secondary structure, and positioned adjacent to the substrate phosphate in a tertiary structure model of the ribozyme-substrate complex. The spatial arrangement of phosphorothioate-sensitive sites in RNase P RNA was found to resemble the distribution of analogous positions in the secondary and potential tertiary structures of other large catalytic RNAs.

Base Sequence↗
PubMed1995

Thermotoga subterranea sp. nov., a new thermophilic bacterium isolated from a continental oil reservoir.

A thermophilic, strictly anaerobic bacterium, designated strain SL1, was isolated from a deep, continental oil reservoir in the East Paris Basin (France). This organism grew between 50 and 75 degrees C, with an optimum at 70 degrees C. It was inhibited by elemental sulfur and was able to reduce cystine and thiosulfate to hydrogen sulfide. The G+C content (40 mol%), the presence of a lipid structure unique to the genus Thermotoga, and the 16S rRNA sequence of strain SL1 indicated that the isolate belongs to the genus Thermotoga. Based on DNA-DNA hybridization, isolate SL1 does not show species-level similarity with the recognized species T. maritima, T. neapolitana, and T. thermarum. Based on this description of strain SL1, we propose the recognition of a new species: Thermotoga subterranea.

Base Composition↗
PubMed1995

Analysis of conserved positions in nuclear RNase P RNA.

Secondary structure models of eubacterial and eukaryotic nuclear RNase P RNA subunits show extensive structural similarities, allowing the identification of highly conserved nucleotide positions and molecular modeling of the enzyme-substrate complex in three dimensions. Based on this information, we present a preliminary tertiary structure model of the yeast nuclear RNase P RNA. In addition, the most conserved positions in the structure have been subjected to sequence randomization, with viable sequence variations identified by selection in vivo and characterized for phenotypic consequences.

Base Sequence↗
PubMed1994

Interaction of the 3'-end of tRNA with ribonuclease P RNA.

Ribonuclease P, which contains a catalytic RNA subunit, cleaves 5' precursor-specific sequences from pre-tRNAs. It was previously shown that the RNase P RNA optimally cleaves substrates which contain the mature, 3'-terminal CCA of tRNA. In order to determine the contributions of those individual 3'-terminal nucleotides to the interaction, pre-tRNAs that have CCA, only CC or C or are without CCA at the 3'-end were synthesized by run-off transcription, tested as substrates for cleavage by RNase P RNA and used in photoaffinity crosslinking experiments to examine contact sites in the ribozyme. In order to generalize the results, analyses were carried out using three different bacterial RNase P RNAs, from Escherichia coli, Bacillus subtilis and Thermotoga maritima. At optimal (Kcat/Km) ionic strength (1 M NH4+/25 mM Mg2+), Km increases incrementally 3- to 10-fold upon stepwise removal of each nucleotide from the 3'-end. At high ionic strength (2 M NH4+/50 mM Mg2+), which suppresses conformational effects, removal of the 3'-terminal A had little effect on Km, indicating that it is not a specific contact. Analysis of the deletion and substitution mutants indicated that the C residues act specially; their contribution to binding energy at high ionic strength (approximately 1 kcal/mol) is consistent with a non-Watson-Crick interaction, possibly irregular triple-strand formation with some component of the RNase P RNA. In agreement with previous studies, we find that the RNase P holoenzyme in vitro does not discriminate between tRNAs containing or lacking CCA. The structural elements of the three RNase P RNAs in proximity to the 3'-end of tRNA were examined by photoaffinity crosslinking. Photoagent-labeled tRNAs with 3'-terminal CCA, only CC or C, or lacking all these nucleotides were covalently conjugated to the three RNase P RNAs by irradiation and the sites of crosslinks were mapped by primer extension. The main crosslink sites are located in a highly conserved loop (probably an irregular helix) that is part of the core of the RNase P RNA secondary structure. The crosslinking results orient the CCA of tRNA with respect to that region of the RNase P RNA.

Bacillus subtilis↗
PubMed1994

Rational design of self-cleaving pre-tRNA-ribonuclease P RNA conjugates.

Ribonuclease P (RNaseP) generates the mature 5' end of tRNAs by removing 5'leader sequences from pre-tRNAs. In vitro, the RNA subunit is sufficient to catalyze this reaction and is therefore a ribozyme. The kinetic analysis of RNase P-mediated catalysis is complicated because product release is normally rate-limiting. Furthermore, the intermolecular nature of the cleavage reaction precludes many applications of in vitro selection schemes to the analysis of RNaseP. To examine and manipulate the RNase P function more effectively, we designed a pair of ribozymes in which the RNase P RNA is covalently linked to a pre-tRNA substrate. To facilitate intramolecular cleavage, pre-tRNA molecules were tethered to circulatory permuted RNaseP RNA molecules at nucleotides implicated in substrate binding. These "active-site-tethered" pre-tRNA-RNaseP RNA conjugates undergo accurate and efficient self-cleavage in vitro, with first-order reaction rates equivalent to the rate of the chemical step of the native RNase P reaction. Unlike most ribozymes, RNase P recognizes its substrate through tertiary RNA-RNA interactions, rather than through extensive Watson-Crick base-pairing. However, the development of the active-site-tethered conjugates has led us to create a sequence-specific endonuclease, termed Endo.P. In the Endo.P configuration, the 3'half of the pre-tRNA acceptor stem binds exogenous RNA substrates via Watson-Crick base-pairing; the bound substrate is subsequently cleaved at the predicted site. The demonstration of sequence-specific cleavage by Endo.P expands the potential of RNase P and its derivatives as reagents in gene therapy.

Base Sequence↗
PubMed1994

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.

Bacillus subtilis↗
PubMed1994

Use of photoaffinity crosslinking and molecular modeling to analyze the global architecture of ribonuclease P RNA.

Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein containing a catalytic RNA. The secondary structure of this ribozyme is well established, but comparatively little is understood about its 3-D structure. In this analysis, orientation and distance constraints between elements within the Escherichia coli RNase P RNA-pre-tRNA complex were determined by intra- and intermolecular crosslinking experiments. A molecular mechanics-based RNA structure refinement protocol was used to incorporate the distance constraints indicated by crosslinking, along with the known secondary structure of RNase P RNA and the tertiary structure of tRNA, into molecular models. Seven different structures that satisfy the constraints equally well were generated and compared by superposition to estimate helix positions and orientations. Manual refinement within the range of conformations indicated by the molecular mechanics analysis was used to derive a model of RNase P RNA with bound substrate pre-tRNA that is consistent with the crosslinking results and the available phylogenetic comparisons.

Affinity Labels↗