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.

In vitro synthesis of an infectious mutant RNA with a normal RNA replicase.

When purified Qbeta-RNA replicase is presented alternately with two genetically different Qbeta-RNA molecules, the RNA synthesized is identical to the initiating template. The results establish that the RNA is the instructive agent in the replicative process and hence that it satisfies the operational definition of a self-duplicating entity. The data also eliminate alternative explanations which do not involve self-propagation of the input RNA. An opportunity is now provided for studying the genetics and evolution of a self-duplicating nucleic acid molecule under conditions permitting detailed control of environmental parameters and chemical components.

Ribonuclease P catalysis differs from ribosomal RNA self-splicing.

Two RNA-catalyzed reactions have been described, the Tetrahymena self-splicing ribosomal RNA and ribonuclease P. The Tetrahymena self-splicing reaction proceeds through a transesterification cascade that is dependent upon nucleophilic attacks by ribose 3'-OH groups. Periodate oxidation of the catalytic (or substrate) RNA, which destroys the nucleophilicity of RNA 3' termini, did not inhibit ribonuclease P activity. Thus, catalysis by ribonuclease P differs from the self-splicing reaction.

Circularly permuted tRNAs as specific photoaffinity probes of ribonuclease P RNA structure.

Regions of Escherichia coli ribonuclease P (RNase P) RNA in proximity to a bound transfer RNA (tRNA) substrate were mapped by photoaffinity. A photoaffinity cross-linking reagent was introduced at specific sites in the interior of the native tRNA structure by modification of the 5' ends of circularly permuted tRNAs (cptRNAs). The polymerase chain reaction was used for the production of cptRNA templates. After the amplification of a segment of a tandemly duplicated tRNA gene, the cptRNA gene was transcribed in vitro to produce cptRNA. Modified cptRNAs were cross-linked to RNase P RNA, and the conjugation sites in RNase P RNA were determined by primer extension. These sites occur in phylogenetically conserved structures and sequences and identify regions of the ribozyme that form part of the tRNA binding site. The use of circularly permuted molecules to position specific modifications is applicable to the study of many inter- and intramolecular interactions.

Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells.

Rapid phylogenetic identification of single microbial cells was achieved with a new staining method. Formaldehyde-fixed, intact cells were hybridized with fluorescently labeled oligodeoxynucleotides complementary to 16S ribosomal RNA (rRNA) and viewed by fluorescence microscopy. Because of the abundance of rRNA in cells, the binding of the fluorescent probes to individual cells is readily visualized. Phylogenetic identification is achieved by the use of oligonucleotides (length 17 to 34 nucleotides) that are complementary to phylogenetic group-specific 16S rRNA sequences. Appropriate probes can be composed of oligonucleotide sequences that distinguish between the primary kingdoms (eukaryotes, eubacteria, archaebacteria) and between closely related organisms. The simultaneous use of multiple probes, labeled with different fluorescent dyes, allows the identification of different cell types in the same microscopic field. Quantitative microfluorimetry shows that the amount of an rRNA-specific probe that binds to Escherichia coli varies with the ribosome content and therefore reflects growth rate.

The design and catalytic properties of a simplified ribonuclease P RNA.

Ribonuclease P (RNase P) RNA is the catalytic moiety of the ribonucleoprotein enzyme that removes precursor sequences from the 5' ends of pre-transfer RNAs in eubacteria. Phylogenetic variation according to recently proposed secondary structure models was used to identify structural elements of the RNase P RNA that are dispensable for catalysis. A simplified RNase P RNA that consists only of evolutionarily conserved features was designed, synthesized, and characterized. Although the simplified RNA (Min 1 RNA) is only 263 nucleotides in length, in contrast to the 354 to 417 nucleotides of naturally occurring RNase P RNAs, its specificity of pre-tRNA cleavage is identical to that of the native enzymes. Moreover, the catalytic efficiencies of the Min 1 RNA and the native RNA enzymes are similar. These results focus the search for the catalytic elements of RNase P RNAs to their conserved structure.

Role of the protein moiety of ribonuclease P, a ribonucleoprotein enzyme.

The Bacillus subtilis ribonuclease P consists of a protein and an RNA. At high ionic strength the reaction is protein-independent; the RNA alone is capable of cleaving precursor transfer RNA, but the turnover is slow. Kinetic analyses show that high salt concentrations facilitate substrate binding in the absence of the protein, probably by decreasing the repulsion between the polyanionic enzyme and substrate RNAs, and also slow product release and enzyme turnover. It is proposed that the ribonuclease P protein, which is small and basic, provides a local pool of counter-ions that facilitates substrate binding without interfering with rapid product release.

Analysis of hydrothermal vent-associated symbionts by ribosomal RNA sequences.

Ribosomal RNA (rRNA) sequences were used to establish the phylogenetic affiliations of symbioses in which prokaryotes appear to confer sulfur-based chemoautotrophy on their invertebrate hosts. Two submarine hydrothermal vent animals, the vestimentiferan tube worm Riftia pachyptila and the clam Calyptogena magnifica, and a tidal-flat bivalve, Solemya velum, were inspected. 5S rRNA's were extracted from symbiont-bearing tissues, separated into unique forms, and their nucleotide sequences determined and related to other 5S rRNA's in a phylogenetic tree analysis. The prokaryotic symbionts are related to one another and affiliated with the same narrow phylogenetic grouping as Escherichia coli and Pseudomonas aeruginosa. The sequence comparisons suggest that Riftia is more closely related to the bivalves than their current taxonomic status would suggest.

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.

Molecular phylogeny of the animal kingdom.

A rapid sequencing method for ribosomal RNA was applied to the resolution of evolutionary relationships among Metazoa. Representatives of 22 classes in 10 animal phyla were used to infer phylogenetic relationships, based on evolutionary distances determined from pairwise comparisons of the 18S ribosomal RNA sequences. The classical Eumetazoa are divided into two groups. Cnidarians arose from a protist ancestry different from the second group, the Bilateria. Within the Bilateria, an early split gave rise to Platyhelminthes (flatworms) and the coelomate lineage. Coelomates are thus monophyletic, and they radiated rapidly into four groups: chordates, echinoderms, arthropods, and eucoelomate protostomes.

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.

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.

Phylogenetic comparative analysis and the secondary structure of ribonuclease P RNA--a review.

The most incisive a priori approach to inferring the higher order structure of large RNAs has proven to be the use of phylogenetic comparisons. This article provides guidelines to the method, using as an illustration the elucidation of the secondary structure of the catalytic RNA subunit of ribonuclease P (RNase P). The resultant structure is compared to the possibilities that are predicted thermodynamically for the RNase P RNA sequences of nine eubacteria.

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.