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.

The ribosomal RNAs (rRNA) of all cells are acted upon by several RNA processing nucleases and nucleotide-modifying enzymes during the formation of the mature ribosome. The general requirement of the terminal maturases for RNP substrates is best evidenced by the fact that the immediate precursors of the mature rRNAs accumulate in cells treated with inhibitors of protein synthesis. The proteins required for the formation of productive substrates for the terminal rRNA maturases probably are generally ribosomal proteins, known to associate with the nascent rRNAs during transcription. The terminal rRNA maturases are rare: each B. subtilis cell probably contains no more than about I00 molecules of RNase M5. The extensive purification of rare enzymes such as RNase M5 inevitably results in low solution concentrations of protein, a condition that is considered to predispose enzymes to denaturation and loss of activity. Most of the reported studies of RNase M5 used enzyme at this level of purity. Preparations are greatly enriched in RNase M5 activity and free of other nucleases.

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.

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.

The 16S rRNAs from the bacterial endosymbionts of six marine invertebrates from diverse environments were isolated and partially sequenced. These symbionts included the trophosome symbiont of Riftia pachyptila, the gill symbionts of Calyptogena magnifica and Bathymodiolus thermophilus (from deep-sea hydrothermal vents), and the gill symbionts of Lucinoma annulata, Lucinoma aequizonata, and Codakia orbicularis (from relatively shallow coastal environments). Only one type of bacterial 16S rRNA was detected in each symbiosis. Using nucleotide sequence comparisons, we showed that each of the bacterial symbionts is distinct from the others and that all fall within a limited domain of the gamma subdivision of the purple bacteria (one of the major eubacterial divisions previously defined by 16S rRNA analysis [C. R. Woese, Microbiol. Rev. 51: 221-271, 1987]). Two host specimens were analyzed in five of the symbioses; in each case, identical bacterial rRNA sequences were obtained from conspecific host specimens. These data indicate that the symbioses examined are species specific and that the symbiont species are unique to and invariant within their respective host species.

The16SrRNAsfromthebacterial endosymbionts ofsixmarine invertebrates fromdiverse environments wereisolated andpartially sequenced. These symbionts included thetrophosome symbiont ofRiftia pachyptila, thegill symbionts ofCalyptogena magnffica andBathymodiolus thermophilus (from deep-sea hydrothermal vents), andthegill symbionts ofLucinoma annulata, Lucinoma aequizonata, andCodakia orbicularis (from relatively shallow coastal environments). Onlyonetype ofbacterial 16SrRNAwasdetected ineachsymbiosis. Using nucleotide sequence comparisons, weshowed that eachofthebacterial symbionts isdistinct fromthe others andthatallfall within alimited domain ofthegammasubdivision ofthepurple bacteria (one ofthe majoreubacterial divisions previously defined by16SrRNA analysis (C.R.Woese,Microbiol. Rev. 51:221-271, 1987)). Twohost specimens wereanalyzed infive ofthesymbioses; ineachcase, identical bacterial rRNAsequences wereobtained fromconspecific hostspecimens. Thesedataindicate thatthesymbioses examined arespecies specific andthat thesymbiont species areunique toandinvariant within their respective hostspecies.

Ribonuclease P (RNase P) is the enzyme responsible for removing the 5′ precursor segments from tRNA during its maturation. RNase P is particularly interesting because its catalytic element is an RNA, not a protein (Guerrier-Takada et al. 1983; Gardiner et al. 1985). Although the recognition of RNase P as a catalytic RNA was preceded by the discovery of a self-splicing intron in some Tetrahymena 26S rRNA precursors (Kruger et al. 1982), the RNase P RNA differs in an important way: It engages in intermolecular reactions. In contrast, the self-splicing intron activity in vivo is a series of intramolecular rearrangements that collectively result in the excision of the intron and the ligation of the flanking exons (for review, see Cech 1985). RNase P therefore offers not only a model for RNA catalytic mechanisms, but also a system for exploring the nature of specific RNA-RNA recognition that almost certainly goes beyond the...

The gene defining the catalytic RNA component of RNase P in Bacillus subtilis 168 was cloned into bacteriophage lambda and plasmid vectors. The nucleotide sequence of the gene and its surroundings was determined from the cloned DNA and by directly sequencing or reverse transcribing the RNase P RNA. The B. subtilis RNase P RNA sequence (400-401 nucleotides) is remarkably different from that of Escherichia coli (377 nucleotides) (Reed, R. E., Baer, M. F., Guerrier-Takada, C., Donis-Keller, H., and Altman, S. (1982) Cell 30, 627-636; Sakamoto, H., Kimura, N., Nagawa, F., and Shimura, Y. (1983) Nucleic Acids Res. 11, 8237-8251). At best the two are less than 50% similar in sequence. To verify that the RNase P RNA gene was analyzed, a modified, putative gene was cloned adjacent to a bacteriophage T7 promoter and various transcripts were tested for RNase P activity. The intact gene transcript, but not fragments, showed full activity. Full catalytic activity was restored upon mixing the fragments. The extensive differences between the B. subtilis and E. coli RNase P RNAs precluded full covariance analysis of secondary structure, but phylogenetically consistent foldings for portions of both molecules could be derived.

Although the applicability of small subunit ribosomal RNA (16S rRNA) sequences for bacterial classification is now well accepted, the general use of these molecules has been hindered by the technical difficulty of obtaining their sequences. A protocol is described for rapidly generating large blocks of 16S rRNA sequence data without isolation of the 16S rRNA or cloning of its gene. The 16S rRNA in bulk cellular RNA preparations is selectively targeted for dideoxynucleotide-terminated sequencing by using reverse transcriptase and synthetic oligodeoxynucleotide primers complementary to universally conserved 16S rRNA sequences. Three particularly useful priming sites, which provide access to the three major 16S rRNA structural domains, routinely yield 800-1000 nucleotides of 16S rRNA sequence. The method is evaluated with respect to accuracy, sensitivity to modified nucleotides in the template RNA, and phylogenetic usefulness, by examination of several 16S rRNAs whose gene sequences are known. The relative simplicity of this approach should facilitate a rapid expansion of the 16S rRNA sequence collection available for phylogenetic analyses.

We have tested a putative base-paired interaction between the conserved GT psi C sequence of tRNA and the conserved GAAC47 sequence of 5 S ribosomal RNA by in vitro protein synthesis using ribosomes containing deletions in this region of 5 S rRNA. Ribosomes reconstituted with 5 S rRNA possessing a single break between residues 41 and 42, deletion of residues 42-46, or deletion of residues 42-52 were tested for their ability to translate phage MS2 RNA. Initiator tRNA binding, aminoacyl-tRNA binding, ppGpp synthesis, and miscoding were also tested. All of the measured functions could be carried out by ribosomes carrying the deleted 5 S rRNAs. The sizes and relative amounts of the polypeptides synthesized by MS2 RNA-programmed ribosomes were identical whether or not the 5 S RNA contained deletions. Aminoacyl-tRNA binding and miscoding were essentially unaffected. Significant reduction in ApUpG (but not poly(A,U,G) or MS2 RNA)-directed fMet-tRNA binding and ppGpp synthesis were observed, particularly in the case of the larger (residues 42-52) deletion. We conclude that if tRNA and 5 S rRNA interact in this fashion, it is not an obligatory step in protein synthesis.

The Bacillus subtilis RNase M5 complex, responsible for the terminal maturation of 5 S rRNA, includes two proteins. One of these proteins is ribosomal protein BL16 (equivalent to Escherichia coli EL18); the other, the alpha component, is required for catalysis. The RNase M5 alpha component has been purified in bulk extensively, and the active polypeptide (Mr approximately 24,000) identified following polyacrylamide gel electrophoresis. Reaction conditions (20-30% dimethyl sulfoxide) are reported which render RNase M5 activity independent of ribosomal protein BL16. This proves that alpha indeed is the catalytic element, the actual RNase M5, which normally attacks a ribonucleoprotein substrate consisting of protein BL16 in complex with the 5 S rRNA precursor. Kinetic analyses of the BL16-dependent and independent reactions suggest that any alpha-BL16 association contributes little to the energetics of the alpha-ribonucleoprotein substrate interaction. It is postulated that the BL16 protein serves as a scaffold, to lock the precursor mRNA into a conformation recognizable by the nuclease.

We have tested a putative base-paired interaction between the conserved GTGC sequence of tRNA and the conserved GAAC,, sequence of 5 S ribosomal RNA by in vitro protein synthesis using ribosomes containing deletions in this region of 5 S rRNA.Ribosomes reconstituted with 5 S rRNA possessing a single break between residues 41 and 42, deletion of residues 42-46, or deletion of residues 42-52 were tested for their ability to translate phage MS2 RNA.Initiator tRNA binding, aminoacyl-tRNA binding, ppGpp synthesis, and miscoding were also tested.All of the measured

The Bacillus subtilis RNase M5 activity, responsible for the endonucleolytic maturation of 5 S rRNA, requires two proteins, alpha and beta. The beta component has been purified to homogeneity and shown to correspond to ribosomal protein BL16. The BL16 protein evidently corresponds functionally to Escherichia coli ribosomal protein EL18, as that latter protein also will complement the B. subtilis alpha protein in the RNase M5 reaction. A filter binding assay for the formation of B. subtilis 5 S rRNA-protein complexes was characterized and used to evaluate the association of BL16 protein with some RNAs. A native precursor of 5 S rRNA, containing extra sequences at both termini of the mature domain, binds the ribosomal protein no better than the mature 5 S rRNA; the precursor sequences do not facilitate that interaction. A model is considered in which the precursor segments facilitate, by refolding, the dissociation of processing products prior to the RNase M5 step. Electrostatic versus nonelectrostatic contributions to the BL16-5 S rRNA complex formation were inspected by analyzing variation in apparent association constants as a function of ionic strength. Electrostatic interactions were seen to contribute approximately 65% to the overall binding energy.

The notion that tRNA and 5S rRNA interact through evolutionarily conserved complementary sequences has been tested by nucleolytic modification of the 5S rRNA, using the modified rRNA to reconstitute the large ribosomal subunit, and assaying for poly(uridylic acid)-directed polyphenylalanine synthesis. The 5S rRNA sequence C-G-A-A (residues 43-46) and several residues surrounding it are not essential for protein synthesis.

Several boronate-containing supports for the separation of oligo- or polyribonucleotides, on the basis of their content vs lack of cis-diol groups, have been described in the literature; boronate cellulose (DBAE-cellulose) and boronate polyacrylamide are available commercially. Boronate cellulose binds macromolecular RNA, but often is not useful for trace amounts of material because of nonspecific adsorption. We have found that boronate polyacrylamide and boronate agarose (not yet commercially available) both display very low levels of nonspecific adsorption. Boronate polyacrylamide specifically binds cis-diol-containing mononucleotides or short oligonucleotides, but does not retain polyribonucleotides sufficiently tenaciously for preparative use. In contrast, boronate agarose proved suitable for the fractionation of trace amounts of macromolecular RNA, but not for mononucleotides.

As is evident from several other papers in this volume, considerable information regarding the posttranscriptional processing of tRNA is accumulating. Although most RNA molecules, in both prokaryotes and eukaryotes, are the products of more or less extensive posttranscriptional metabolism, detailed studies of these processes have been possible only with tRNA and 5S rRNA of Bacillus subtilis. Although not directly involved with tRNA, studies of the processing of 5S rRNA are relevant in this area of study in that much of this methodology is immediately applicable to the study of the maturation of the precursors of tRNA. Moreover, there is the common goal of understanding the nature of protein-polynucleotide interactions. In brief, we have isolated the endonuclease responsible for the terminal maturation of 5S rRNA in B. subtilis and devised procedures for altering the precursor RNA substrate to explore the polynucleotide recognition elements utilized by this highly specific enzyme. THE MATURATION OF B. SUBTILIS 5S rRNA Because of its simple structure (about 120 nucleotides long), 5S rRNA is useful for exploring the details of posttranscriptional RNA metabolism. At the outset of these studies, we felt that the 5S rRNA of Escherichia coli was not likely to be a suitable model, since the immediate precursor of 5S rRNA in this organism is maximally only 3 nucleotides larger than the mature form (Monier et al. 1970) and such limited precursor-specific length might not be of functional significance. We therefore undertook a search for an organism utilizing more complex 5S rRNA metabolism than that...

RNase M5 of Bacillus subtilis specifically cleaves a 179-nucleotide precursor 5S rRNA to yield mature 5S rRNA (116 nucleotides) and two fragments derived from the termini. Possible recognition elements for RNase M5 within the precursor structure include nucleotide sequences arranged with 2-fold rotational and translational symmetry about the substrate bonds. We have used bacteriophage T4 RNA ligase to construct, from synthetic oligonucleotides and mature or precursor 5S rRNA fragments, test substrates lacking these symmetry elements. The susceptibilities of the artificial substrates to RNase M5 demonstrate that the symmetrically arranged sequences are not used in the RNase M5 interaction with the precursor. Additionally, the synthetic protocols permitted the invention of an acid-soluble assay for RNase M5 and, potentially, other specific endoribonucleases.

Data are presented on the partial purification and properties of a 5 S ribosomal RNA maturation nuclease, termed RNase M5, from Bacillus subtillis 168. RNase M5 specifically cleaves 21 and 42 nucleotides, respectively, from the 5' and 3' termini of a 5 S rRNA precursor to yield the mature (116 nucleotides) 5 S rRNA. The cleavage is endonucleolytic with the formation of 5'-phosphoryl and 3'-hydroxyl groups. Enzyme action requires divalent cations, which may be furnished by either certain metals or by polyamines. The activity is separable into two components both of which are required for activity. It appears that the same nuclease excises the 5'- and 3'-terminal segments since preparations lose the capacity to modify the two termini with an identical first order thermal decay rate. Certain features of the rRNA precursor which may be involved in cognitive interaction with RNase M5 are discussed.