The x-ray crystal structure of a 417-nt ribonuclease P RNA from Bacillus stearothermophilus was solved to 3.3-Å 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.ribozyme ͉ RNA crystallography ͉ tRNA processing R Nase P catalyzes hydrolysis of a phosphodiester bond in precursors of transfer RNA (tRNA) to form the 5Ј-phosphorylated mature tRNA with the release of a 5Ј-precursor fragment (1, 2). RNase P homologs occur in all organisms, and the cellular RNase P always is a ribonucleoprotein that consists of one large RNA and one or more protein component. In bacteria, RNase P is typically comprised of a 350-to 400-nt RNA and one Ϸ120-aa basic protein. Although both RNA and protein components are necessary for cell viability, in vitro at high salt concentrations, bacterial RNase P RNA can act as a catalyst independently of protein (3). Bacterial RNase P is a ribozyme, an RNA-based enzyme.Knowledge of the structure of RNase P RNA is essential for understanding its function, and structure has been the focus of numerous studies of the RNA. Phylogenetic comparative analyses of RNase P RNA sequences have established the secondary and some tertiary structure of the RNA in a broad diversity of organisms (4-8). Photochemical crosslinking studies provided structural information to orient the helical elements and identified nucleotides associated with the active site of the RNA (9, 10). There are two major structural types of bacterial RNase P RNA, A (ancestral) and B (Bacillus), which differ in a number of structural elements attached to a homologous conserved structure. About two-thirds of any bacterial RNase P RNA is shown by sequence covariations to be involved in Watson-Crick base-pairing interactions, but the interactions that form the global structure have been speculative.To gain a better understanding of bacterial RNase P, we crystallized and solved the structure of a 417-nt B-type RNase P RNA from Bacillus stearothermophilus, a moderately thermophilic, low GϩC Gram-positive bacterium. Although the structure does not yet explain the chemical mechanism of catalysis, it is in agreement with a wealth of available biochemical and comparative data, and it provides a structural context for the chemically active site of this ribozyme. Materials and MethodsRNA Purification, Crystallization, and Data Collection. As detailed in supporting information, which is published on the PNAS web site, RNA was transcribed in vitro with T7 phage RNA ...

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.