Thermostable RNase P RNAs lacking P18 identified in the Aquificales

The RNase P RNA (rnpB) and protein (rnpA) genes were identified in the two Aquificales Sulfurihydrogenibium azorense and Persephonella marina. In contrast, neither of the two genes has been found in the sequenced genome of their close relative, Aquifex aeolicus. As in most bacteria, the rnpA genes of S. azorense and P. marina are preceded by the rpmH gene coding for ribosomal protein L34. This genetic region, including several genes up- and downstream of rpmH, is uniquely conserved among all three Aquificales strains, except that rnpA is missing in A. aeolicus. The RNase P RNAs (P RNAs) of S. azorense and P. marina are active catalysts that can be activated by heterologous bacterial P proteins at low salt. Although the two P RNAs lack helix P18 and thus one of the three major interdomain tertiary contacts, they are more thermostable than Escherichia coli P RNA and require higher temperatures for proper folding. Related to their thermostability, both RNAs include a subset of structural idiosyncrasies in their S domains, which were recently demonstrated to determine the folding properties of the thermostable S domain of Thermus thermophilus P RNA. Unlike 16S rRNA phylogeny that has placed the Aquificales as the deepest lineage of the bacterial phylogenetic tree, RNase P RNA-based phylogeny groups S. azorense and P. marina with the green sulfur, cyanobacterial, and delta/epsilon proteobacterial branches.     

Sequences encoding the protein and RNA components of ribonuclease P from Streptomyces bikiniensis var. zorbonensis.

The genes encoding the RNA (rnpB) and protein (rnpA) subunits of ribonuclease P (RNase P) of Streptomyces bikiniensis var. zorbonensis have been cloned by complementing the temperature-sensitive growth phenotype of Escherichia coli strains that carry mutations in these genes. The rnpB sequence of S. bikiniensis includes new covariations that lead to refinement of the previous secondary structure models for RNase P RNAs. The deduced amino acid sequence of S. bikiniensis RNase P is conserved with that of other known RNase P proteins only to a limited extent. Immediately upstream from rnpA is an open reading frame that codes for the highly conserved ribosomal protein, L34. This same gene arrangement occurs in all bacteria studied to date.     

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.     

YbxF and YlxQ are bacterial homologs of L7Ae and bind K-turns but not K-loops

The archaeal protein L7Ae and eukaryotic homologs such as L30e and 15.5kD comprise the best characterized family of K-turn-binding proteins. K-turns are an RNA motif comprised of a bulge flanked by canonical and noncanonical helices. They are widespread in cellular RNAs, including bacterial gene-regulatory RNAs such as the c-di-GMP-II, lysine, and SAM-I riboswitches, and the T-box. The existence in bacteria of K-turn-binding proteins of the L7Ae family has not been proven, although two hypothetical proteins, YbxF and YlxQ, have been proposed to be L7Ae homologs based on sequence conservation. Using purified, recombinant proteins, we show that Bacillus subtilis YbxF and YlxQ bind K-turns (K(d) ~270 nM and ~2300 nM, respectively). Crystallographic structure determination demonstrates that both YbxF and YlxQ adopt the same overall fold as L7Ae. Unlike the latter, neither bacterial protein recognizes K-loops, a structural motif that lacks the canonical helix of the K-turn. This property is shared between the bacterial and eukaryal family members. Comparison of our structure of YbxF in complex with the K-turn of the SAM-I riboswitch and previously determined structures of archaeal and eukaryal homologs bound to RNA indicates that L7Ae approaches the K-turn at a unique angle, which results in a considerably larger RNA-protein interface dominated by interactions with the noncanonical helix of the K-turn. Thus, the inability of the bacterial and eukaryal L7Ae homologs to bind K-loops probably results from their reliance on interactions with the canonical helix. The biological functions of YbxF and YlxQ remain to be determined.     

Protein activation of a ribozyme: the role of bacterial RNase P protein

Bacterial ribonuclease P (RNase P) belongs to a class of enzymes that utilize both RNAs and proteins to perform essential cellular functions. The bacterial RNase P protein is required to activate bacterial RNase P RNA in vivo, but previous studies have yielded contradictory conclusions regarding its specific functions. Here, we use biochemical and biophysical techniques to examine all of the proposed functions of the protein in both Escherichia coli and Bacillus subtilis RNase P. We demonstrate that the E. coli protein, but not the B. subtilis protein, stabilizes the global structure of RNase P RNA, although both proteins influence holoenzyme dimer formation and precursor tRNA recognition to different extents. By comparing each protein in complex with its cognate and noncognate RNA, we show that differences between the two types of holoenzymes reside primarily in the RNA and not the protein components of each. Our results reconcile previous contradictory conclusions regarding the role of the protein and support a model where the protein activates local RNA structures that manifest multiple holoenzyme properties.     

Isolation of RNase H genes that are essential for growth of Bacillus subtilis 168

Two genes encoding functional RNase H (EC 3.1.26.4) were isolated from a gram-positive bacterium, Bacillus subtilis 168. Two DNA clones exhibiting RNase H activities both in vivo and in vitro were obtained from a B. subtilis DNA library. One (28.2 kDa) revealed high similarity to Escherichia coli RNase HII, encoded by the rnhB gene. The other (33.9 kDa) was designated rnhC and encodes B. subtilis RNase HIII. The B. subtilis genome has an rnhA homologue, the product of which has not yet shown RNase H activity. Analyses of all three B. subtilis genes revealed that rnhB and rnhC cannot be simultaneously inactivated. This observation indicated that in B. subtilis both the rnhB and rnhC products are involved in certain essential cellular processes that are different from those suggested by E. coli rnh mutation studies. Sequence conservation between the rnhB and rnhC genes implies that both originated from a single ancestral RNase H gene. The roles of bacterial RNase H may be indicated by the single rnhC homologue in the small genome of Mycoplasma species.     

Rpp14 and Rpp29, two protein subunits of human ribonuclease P

In HeLa cells, the tRNA processing enzyme ribonuclease P (RNase P) consists of an RNA molecule associated with at least eight protein subunits, hPop1, Rpp14, Rpp20, Rpp25, Rpp29, Rpp30, Rpp38, and Rpp40. Five of these proteins (hPop1p, Rpp20, Rpp30, Rpp38, and Rpp40) have been partially characterized. Here we report on the cDNA cloning and immunobiochemical analysis of Rpp14 and Rpp29. Polyclonal rabbit antibodies raised against recombinant Rpp14 and Rpp29 recognize their corresponding antigens in HeLa cells and precipitate catalytically active RNase P. Rpp29 shows 23% identity with Pop4p, a subunit of yeast nuclear RNase P and the ribosomal RNA processing enzyme RNase MRP. Rpp14, by contrast, exhibits no significant homology to any known yeast gene. Thus, human RNase P differs in the details of its protein composition, and perhaps in the functions of some of these proteins, from the yeast enzyme.     

Structure and function of eukaryotic Ribonuclease P RNA

Ribonuclease P (RNase P) is the ribonucleoprotein endonuclease that processes the 5' ends of precursor tRNAs. Bacterial and eukaryal RNase P RNAs had the same primordial ancestor; however, they were molded differently by evolution. RNase P RNAs of eukaryotes, in contrast to bacterial RNAs, are not catalytically active in vitro without proteins. By comparing the bacterial and eukaryal RNAs, we can begin to understand the transitions made between the RNA and protein-dominated worlds. We report, based on crosslinking studies, that eukaryal RNAs, although catalytically inactive alone, fold into functional forms and specifically bind tRNA even in the absence of proteins. Based on the crosslinking results and crystal structures of bacterial RNAs, we develop a tertiary structure model of the eukaryal RNase P RNA. The eukaryal RNA contains a core structure similar to the bacterial RNA but lacks specific features that in bacterial RNAs contribute to catalysis and global stability of tertiary structure.     

Studies on the mechanism of inhibition of bacterial ribonuclease P by aminoglycoside derivatives

Ribonuclease P (RNase P) is a Mg²⁺-dependent endoribonuclease responsible for the 5′-maturation of transfer RNAs. It is a ribonucleoprotein complex containing an essential RNA and a varying number of protein subunits depending on the source: at least one, four and nine in Bacteria, Archaea and Eukarya, respectively. Since bacterial RNase P is required for viability and differs in structure/subunit composition from its eukaryal counterpart, it is a potential antibacterial target. To elucidate the basis for our previous finding that the hexa-arginine derivative of neomycin B is 500-fold more potent than neomycin B in inhibiting bacterial RNase P, we synthesized hexa-guanidinium and -lysyl conjugates of neomycin B and compared their inhibitory potential. Our studies indicate that side-chain length, flexibility and composition cumulatively account for the inhibitory potency of the aminoglycoside-arginine conjugates (AACs). We also demonstrate that AACs interfere with RNase P function by displacing Mg²⁺ ions. Moreover, our finding that an AAC can discriminate between a bacterial and archaeal (an experimental surrogate for eukaryal) RNase P holoenzyme lends promise to the design of aminoglycoside conjugates as selective inhibitors of bacterial RNase P, especially once the structural differences in RNase P from the three domains of life have been established.     

Interactions of a Pop5/Rpp1 heterodimer with the catalytic domain of RNase MRP

Ribonuclease (RNase) MRP is a multicomponent ribonucleoprotein complex closely related to RNase P. RNase MRP and eukaryotic RNase P share most of their protein components, as well as multiple features of their catalytic RNA moieties, but have distinct substrate specificities. While RNase P is practically universally found in all three domains of life, RNase MRP is essential in eukaryotes. The structural organizations of eukaryotic RNase P and RNase MRP are poorly understood. Here, we show that Pop5 and Rpp1, protein components found in both RNase P and RNase MRP, form a heterodimer that binds directly to the conserved area of the putative catalytic domain of RNase MRP RNA. The Pop5/Rpp1 binding site corresponds to the protein binding site in bacterial RNase P RNA. Structural and evolutionary roles of the Pop5/Rpp1 heterodimer in RNases P and MRP are discussed.     

Heterodimerization regulates RNase MRP/RNase P association, localization, and expression of Rpp20 and Rpp25

Rpp20 and Rpp25 are subunits of the human RNase MRP and RNase P endoribonucleases belonging to the Alba superfamily of nucleic acid binding proteins. These proteins, which bind very strongly to each other, transiently associate with RNase MRP. Here, we show that the Rpp20-Rpp25 heterodimer is resistant to both high concentrations of salt and a nonionic detergent. The interaction of Rpp20 and Rpp25 with the P3 domain of the RNase MRP RNA appeared to be strongly enhanced by their heterodimerization. Coimmunoprecipitation experiments demonstrated that only a single copy of each of these proteins is associated with the RNase MRP and RNase P particles in HEp-2 cells. Both proteins accumulate in the nucleoli, which in case of Rpp20 is strongly dependent on its interaction with Rpp25. Finally, the results of overexpression and knock-down experiments indicate that their expression levels are codependent. Taken together, these data indicate that the Rpp20-Rpp25 heterodimerization regulates their RNA-binding activity, subcellular localization, and expression, which suggests that their interaction is also crucial for their role in RNase MRP/P function.     

By any other name: heterologous replacement of the Escherichia coli RNase P protein subunit has in vivo fitness consequences

Bacterial RNase P is an essential ribonucleoprotein composed of a catalytic RNA component (encoded by the rnpB gene) and an associated protein moiety (encoded by rnpA). We construct a system that allows for the deletion of the essential endogenous rnpA copy and for its simultaneous replacement by a heterologous version of the gene. Using growth rate as a proxy, we explore the effects on fitness of heterologous replacement by increasingly divergent versions of the RNase P protein. All of the heterologs tested complement the loss of the endogenous rnpA gene, suggesting that all existing bacterial versions of the rnpA sequence retain the elements required for functional interaction with the RNase P RNA. All replacements, however, exact a cost on organismal fitness, and particularly on the rate of growth acceleration, defined as the time required to reach maximal growth rate. Our data suggest that the similarity of the heterolog to the endogenous version--whether defined at the sequence, structure or codon usage level--not predict the fitness costs of the replacement. The common assumption that sequence similarity predicts functional similarity requires experimental confirmation and may prove to be an oversimplification.     

An important piece of the RNase P jigsaw solved

RNase P is the ribonucleoprotein enzyme that generates the mature 5' ends of tRNAs throughout all three kingdoms of life. Long known to function as a ribozyme in bacteria and several archaea, it has remained unclear if eukaryal RNase P has entirely lost this RNA-alone catalytic capacity (i.e. the ability to perform catalysis even if the protein part of the enzyme is removed). This controversial debate has now ended after the recent demonstration that eukaryal RNase P also exhibits ribozyme activity.     

Protein-RNA interactions in the subunits of human nuclear RNase P.

A yeast three-hybrid system was employed to analyze interactions in vivo between H1 RNA, the RNA subunit of human nuclear RNase P, and eight of the protein subunits of the enzyme. The genetic analysis indicates that subunits Rpp21, Rpp29, Rpp30, and Rpp38 interact directly with H1 RNA. The results of direct UV crosslinking studies of the purified RNase P holoenzyme confirm the results of the three-hybrid assay.