Solution structure of RNase P RNA.

The ribonucleoprotein enzyme ribonuclease P (RNase P) processes tRNAs by cleavage of precursor-tRNAs. RNase P is a ribozyme: The RNA component catalyzes tRNA maturation in vitro without proteins. Remarkable features of RNase P include multiple turnovers in vivo and ability to process diverse substrates. Structures of the bacterial RNase P, including full-length RNAs and a ternary complex with substrate, have been determined by X-ray crystallography. However, crystal structures of free RNA are significantly different from the ternary complex, and the solution structure of the RNA is unknown. Here, we report solution structures of three phylogenetically distinct bacterial RNase P RNAs from Escherichia coli, Agrobacterium tumefaciens, and Bacillus stearothermophilus, determined using small angle X-ray scattering (SAXS) and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis. A combination of homology modeling, normal mode analysis, and molecular dynamics was used to refine the structural models against the empirical data of these RNAs in solution under the high ionic strength required for catalytic activity.

Mapping metal-binding sites in the catalytic domain of bacterial RNase P RNA.

Ribonuclease P (RNase P) is a ribonucleoprotein enzyme that contains a universally conserved, catalytically active RNA component. RNase P RNA requires divalent metal ions for folding, substrate binding, and catalysis. Despite recent advances in understanding the structure of RNase P RNA, no comprehensive analysis of metal-binding sites has been reported, in part due to the poor crystallization properties of this large RNA. We have developed an abbreviated yet still catalytic construct, Bst P7Delta RNA, which contains the catalytic domain of the bacterial RNase P RNA and has improved crystallization properties. We use this mutant RNA as well as the native RNA to map metal-binding sites in the catalytic core of the bacterial RNase P RNA, by anomalous scattering in diffraction analysis. The results provide insight into the interplay between RNA structure and focalization of metal ions, and a structural basis for some previous biochemical observations with RNase P. We use electrostatic calculations to extract the potential functional significance of these metal-binding sites with respect to binding Mg(2+). The results suggest that with at least one important exception of specific binding, these sites mainly map areas of diffuse association of magnesium ions.

Structural perspective on the activation of RNAse P RNA by protein.

Ribonucleoprotein particles are central to numerous cellular pathways, but their study in vitro is often complicated by heterogeneity and aggregation. We describe a new technique to characterize these complexes trapped as homogeneous species in a nondenaturing gel. Using this technique, in conjunction with phosphorothioate footprinting analysis, we identify the protein-binding site and RNA folding states of ribonuclease P (RNase P), an RNA-based enzyme that, in vivo, requires a protein cofactor to catalyze the 5' maturation of precursor transfer RNA (pre-tRNA). Our results show that the protein binds to a patch of conserved RNA structure adjacent to the active site and influences the conformation of the RNA near the tRNA-binding site. The data are consistent with a role of the protein in substrate recognition and support a new model of the holoenzyme that is based on a recently solved crystal structure of RNase P RNA.

Bacterial RNase P: a new view of an ancient enzyme.

Ribonuclease P (RNase P) is a ubiquitous endonuclease that catalyses the maturation of the 5' end of transfer RNA (tRNA). Although it carries out a biochemically simple reaction, RNase P is a complex ribonucleoprotein particle composed of a single large RNA and at least one protein component. In bacteria and some archaea, the RNA component of RNase P can catalyse tRNA maturation in vitro in the absence of proteins. The discovery of the catalytic activity of the bacterial RNase P RNA triggered numerous mechanistic and biochemical studies of the reactions catalysed by the RNA alone and by the holoenzyme and, in recent years, structures of individual components of the RNase P holoenzyme have been determined. The goal of the present review is to summarize what is known about the bacterial RNase P, and to bring together the recent structural results with extensive earlier biochemical and phylogenetic findings.

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.

Crystal structure of a bacterial ribonuclease P RNA.

The x-ray crystal structure of a 417-nt ribonuclease P RNA from Bacillus stearothermophilus was solved to 3.3-A resolution. This RNA enzyme is constructed from a number of coaxially stacked helical domains joined together by local and long-range interactions. These helical domains are arranged to form a remarkably flat surface, which is implicated by a wealth of biochemical data in the binding and cleavage of the precursors of transfer RNA substrate. Previous photoaffinity crosslinking data are used to position the substrate on the crystal structure and to identify the chemically active site of the ribozyme. This site is located in a highly conserved core structure formed by intricately interlaced long-range interactions between interhelical sequences.

Expression, purification, crystallization and preliminary diffraction analysis of RNase P protein from Thermotoga maritima.

Ribonuclease P (RNase P), the ubiquitous endonuclease that catalyzes maturation of the 5'-end of tRNA in bacteria, is a ribonucleoprotein particle composed of one large RNA and one small protein. Two major structural types of bacterial RNase P RNA have been identified by phylogenetic comparative analysis: the A (ancestral) and B (Bacillus) types. The RNase P protein from Thermotoga maritima, a hyperthermophilic bacterium with an A-type RNase P RNA, has been expressed in Escherichia coli. A purification strategy was developed to obtain a protein preparation suitable for crystallization. Protein crystals suitable for diffraction studies were obtained and characterized.

High-resolution structure of RNase P protein from Thermotoga maritima.

The structure of RNase P protein from the hyperthermophilic bacterium Thermotoga maritima was determined at 1.2-A resolution by using x-ray crystallography. This protein structure is from an ancestral-type RNase P and bears remarkable similarity to the recently determined structures of RNase P proteins from bacteria that have the distinct, Bacillus type of RNase P. These two types of protein span the extent of bacterial RNase P diversity, so the results generalize the structure of the bacterial RNase P protein. The broad phylogenetic conservation of structure and distribution of potential RNA-binding elements in the RNase P proteins indicate that all of these homologous proteins bind to their cognate RNAs primarily by interaction with the phylogenetically conserved core of the RNA. The protein is found to dimerize through an extensive, well-ordered interface. This dimerization may reflect a mechanism of thermal stability of the protein before assembly with the RNA moiety of the holoenzyme.