Alexei V. Kazantsev

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.

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-Å 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.

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.

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.

Chapter 9:A Structural Analysis of Ribonuclease P

The endonucleolytic cleavage of precursor sequences from the 5′-end of transfer RNA (tRNA) precursors to form the mature 5′-end is catalyzed by a remarkably complex and unusual enzyme, ribonuclease P (RNase P)(Figure 9.1).1–4 RNase P is ubiquitous, found in cells from all three domains of life: Bact...