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N R Pace

Publications and source records attributed to N R Pace.

At least 19 recordsLinked to original sources
PubMed1992

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.

Autoradiography↗
PubMed1992

Contributions of phylogenetically variable structural elements to the function of the ribozyme ribonuclease P.

Ribonuclease P (RNase P) is a ribonucleoprotein enzyme which participates in processing precursor tRNAs. The RNA subunit contains the catalytic site and is capable of catalysis in the absence of the protein subunit. RNase P RNAs from various eubacteria consist of a core of conserved sequence and secondary structure which is evolutionarily modified in different organisms by the presence of discrete helical elements at various sites in the RNAs. The variable occurrence of these helical elements suggests that they have no important functional role in the enzyme. The Escherichia coli RNase P RNA contains four such elements. It has been shown that simultaneous deletion of all four of them produces an RNA that is functional but has several significant defects which could arise from general disruption of the RNA or from the loss of element-specific functions. This paper describes a more detailed analysis of the role of the variable elements in E. coli RNase P RNA. Removal of one of the elements had no apparent effect on RNase P activity in vitro. Two other elements are required for correct folding of the RNA: their absence confers a requirement for extremely high monovalent salt concentrations, apparently to reduce intramolecular electrostatic repulsion. The fourth element that was tested participates in a long-range structural interaction (pseudoknot) which contributes to the structural stability of the enzyme and affects substrate binding affinity. In the absence of this helix, the RNA becomes temperature-sensitive, and the KM increases 100-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1992

The varieties of ribonuclease P.

Ribonuclease P is a ribozyme involved in tRNA processing that is present in all cells and organelles that synthesize tRNA. Most of our understanding of ribonuclease P derives from studies of the bacterial enzyme. This enzyme has been characterized biochemically and a secondary structure for the RNA subunit has been proposed. Isolation and characterization of ribonuclease P from diverse Archaea and Eukarya are now modifying and adding to our model of this unusual enzyme. The latter instances of RNase P differ from the bacterial version, but similarities are emerging.

Animals↗
PubMed1992

Differential amplification of rRNA genes by polymerase chain reaction.

The polymerase chain reaction (PCR) is used widely to recover rRNA genes from naturally occurring communities for analysis of population constituents. We have found that this method can result in differential amplification of different rRNA genes. In particular, rDNAs of extremely thermophilic archaebacteria often cannot be amplified by the usual PCR methods. The addition of 5% (wt/vol) acetamide to a PCR mixture containing both archaebacterial and yeast DNA templates minimized nonspecific annealing of the primers and prevented preferential amplification of the yeast small-subunit rRNA genes.

Acetamides↗
PubMed1992

Evolutionary relationships among sulfur- and iron-oxidizing eubacteria.

Some 37 reverse transcriptase, partial 16S rRNA sequences from sulfur- and/or iron-oxidizing eubacteria, including sequences from species of the genera Thiobacillus, Thiothrix, Thiomicrospira, Acidophilium, "Leptospirillum," Thiovulum, and Chlorobium, have been determined. In addition, 16S sequences from a number of unnamed sulfur- and/or iron-oxidizing bacteria from hydrothermal vent sites, from invertebrate-bacterial endosymbioses, and from various mineral recovery operations also have been determined. The majority of sequences place their bacterial donors in one or another of the subdivisions of the Proteobacteria. However, three unnamed facultatively thermophilic iron-oxidizing isolates, Alv, BC, and TH3, are affiliated with the gram-positive division. One H2S-oxidizer, from the genus Thiovulum, is affiliated with Campylobacter, Wolinella, and other genera in what appears to be a new subdivision of the Proteobacteria. Three "Leptospirillum"-helical vibrioid isolates, BU-1, LfLa, and Z-2, exhibit no clear phylum level affiliation at all, other than their strong relationship to each other. A picture is emerging of an evolutionary widespread capacity for sulfur and/or iron oxidation among the eubacteria.

Bacteria↗
PubMed1991

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.

Bacillus subtilis↗
PubMed1991

Structure and evolution of ribonuclease P RNA.

Eubacterial RNase P contains a catalytic RNA that cleaves 5' leader sequences from precursor tRNAs. We review the current understanding of RNase P RNA structure and evolution, from the perspective of phylogenetic comparative analysis.

Bacillus subtilis↗
PubMed1991

Phylogenetic analysis of Aquaspirillum magnetotacticum using polymerase chain reaction-amplified 16S rRNA-specific DNA.

The 16S rRNA gene of the magnetotactic magnetogen Aquaspirillum magnetotacticum MS1 was amplified by a polymerase chain reaction, using two eubacterial consensus oligodeoxynucleotide primers flanking the majority of the 16S rRNA gene, cloned, and sequenced. Phylogenetic analysis revealed that A. magnetotacticum MS1 belongs to the alpha-group of proteobacteria. This assignment offers perspective on the biochemical properties of A. magnetotacticum, since this organism is expected to have the general properties that are common to this phylogenetic group.

Base Sequence↗
PubMed1991

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.

Alcaligenes↗
PubMed1991

Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing.

The phylogenetic diversity of an oligotrophic marine picoplankton community was examined by analyzing the sequences of cloned ribosomal genes. This strategy does not rely on cultivation of the resident microorganisms. Bulk genomic DNA was isolated from picoplankton collected in the north central Pacific Ocean by tangential flow filtration. The mixed-population DNA was fragmented, size fractionated, and cloned into bacteriophage lambda. Thirty-eight clones containing 16S rRNA genes were identified in a screen of 3.2 x 10(4) recombinant phage, and portions of the rRNA gene were amplified by polymerase chain reaction and sequenced. The resulting sequences were used to establish the identities of the picoplankton by comparison with an established data base of rRNA sequences. Fifteen unique eubacterial sequences were obtained, including four from cyanobacteria and eleven from proteobacteria. A single eucaryote related to dinoflagellates was identified; no archaebacterial sequences were detected. The cyanobacterial sequences are all closely related to sequences from cultivated marine Synechococcus strains and with cyanobacterial sequences obtained from the Atlantic Ocean (Sargasso Sea). Several sequences were related to common marine isolates of the gamma subdivision of proteobacteria. In addition to sequences closely related to those of described bacteria, sequences were obtained from two phylogenetic groups of organisms that are not closely related to any known rRNA sequences from cultivated organisms. Both of these novel phylogenetic clusters are proteobacteria, one group within the alpha subdivision and the other distinct from known proteobacterial subdivisions. The rRNA sequences of the alpha-related group are nearly identical to those of some Sargasso Sea picoplankton, suggesting a global distribution of these organisms.

Animals↗
PubMed1990

Characterization of ribonuclease P from the archaebacterium Sulfolobus solfataricus.

Ribonuclease P is the endonuclease that removes the leader fragments from the 5'-ends of precursor tRNAs. The enzyme isolated from eubacteria contains a catalytic RNA subunit. RNAs also copurify with eukaryotic RNase P, although catalysis by those RNAs has not been demonstrated. This paper reports the isolation and characterization of ribonuclease P from the thermoacidophilic archaebacterium Sulfolobus solfataricus. Archaebacteria are a primary evolutionary lineage, distinct from both eukaryotes and eubacteria. Ribonuclease P of S. solfataricus has reaction component requirements and a Km for substrate tRNA (2.5 X 10(-7) M) that are roughly similar to those reported for eubacterial and eukaryotic ribonuclease P. The temperature optimum for the reaction is 77 degrees C, reflecting the thermophilic character of the organism. The enzyme activity is not affected by treatment with micrococcal nuclease, suggesting that there is no RNA subunit or that it is protected from nuclease action. The density of the enzyme in cesium sulfate equilibrium density gradients is 1.27 g/ml, which is similar to that of protein. However, several RNAs between 200 and 400 nucleotides in size copurify with the enzyme activity on the density gradients, and one of them remains after micrococcal nuclease treatment. These properties of the S. solfataricus enzyme are compared with those of ribonuclease P from eukaryotes and eubacteria.

Archaea↗