Importance of RNA-protein interactions in bacterial ribonuclease P structure and catalysis

Ribonuclease P (RNase P) is a ribonucleoprotein (RNP) complex that catalyzes the metal-dependent maturation of the 5' end of precursor tRNAs (pre-tRNAs) in all organisms. RNase P is comprised of a catalytic RNA (P RNA), and at least one essential protein (P protein). Although P RNA is the catalytic subunit of the enzyme and is active in the absence of P protein under high salt concentrations in vitro, the protein is still required for enzyme activity in vivo. Therefore, the function of the P protein and how it interacts with both P RNA and pre-tRNA have been the focus of much ongoing research. RNA-protein interactions in RNase P serve a number of critical roles in the RNP including stabilizing the structure, and enhancing the affinity for substrates and metal ions. This review examines the role of RNA-protein interactions in bacterial RNase P from both structural and mechanistic perspectives.     

Molecular modeling of the three-dimensional structure of the bacterial RNase P holoenzyme

Bacterial ribonuclease P (RNase P), an enzyme involved in tRNA maturation, consists of a catalytic RNA subunit and a protein cofactor. Comparative phylogenetic analysis and molecular modeling have been employed to derive secondary and tertiary structure models of the RNA subunits from Escherichia coli (type A) and Bacillus subtilis (type B) RNase P. The tertiary structure of the protein subunit of B.subtilis and Staphylococcus aureus RNase P has recently been determined. However, an understanding of the structure of the RNase P holoenzyme (i.e. the ribonucleoprotein complex) is lacking. We have now used an EDTA-Fe-based footprinting approach to generate information about RNA-protein contact sites in E.coli RNase P. The footprinting data, together with results from other biochemical and biophysical studies, have furnished distance constraints, which in turn have enabled us to build three-dimensional models of both type A and B versions of the bacterial RNase P holoenzyme in the absence and presence of its precursor tRNA substrate. These models are consistent with results from previous studies and provide both structural and mechanistic insights into the functioning of this unique catalytic RNP complex.     

A protein-only RNase P in human mitochondria

In bacteria, archaea, and the eukaryote nucleus, the endonuclease ribonuclease P (RNase P) is composed of a catalytic RNA that is assisted by protein subunits. Holzmann et al. (2008) now provide evidence that the human mitochondrial RNase P is an entirely protein-based enzyme.     

Characterization of conserved sequence elements in eukaryotic RNase P RNA reveals roles in holoenzyme assembly and tRNA processing

RNase P is a ubiquitous endoribonuclease responsible for cleavage of the 5' leader of precursor tRNAs (pre-tRNAs). Although the protein composition of RNase P holoenzymes varies significantly among Bacteria, Archaea, and Eukarya, the holoenzymes have essential RNA subunits with several sequences and structural features that are common to all three kingdoms of life. Additional structural elements of the RNA subunits have been found that are conserved in eukaryotes, but not in bacteria, and might have functions specifically required by the more complex eukaryotic holoenzymes. In this study, we have mutated four eukaryotic-specific conserved regions in Saccharomyces cerevisiae nuclear RNase P RNA and characterized the effects of the mutations on cell growth, enzyme function, and biogenesis of RNase P. RNase P with mutations in each of the four regions tested is sufficiently functional to support life although growth of the resulting yeast strains was compromised to varying extents. Further analysis revealed that mutations in three different regions cause differential defects in holoenzyme assembly, localization, and pre-tRNA processing in vivo and in vitro. These data suggest that most, but not all, eukaryotic-specific conserved regions of RNase P RNA are important for the maturation and function of the holoenzyme.     

An essential protein-binding domain of nuclear RNase P RNA

Eukaryotic RNase P and RNase MRP are endoribonucleases composed of RNA and protein subunits. The RNA subunits of each enzyme share substantial secondary structural features, and most of the protein subunits are shared between the two. One of the conserved RNA subdomains, designated P3, has previously been shown to be required for nucleolar localization. Phylogenetic sequence analysis suggests that the P3 domain interacts with one of the proteins common to RNase P and RNase MRP, a conclusion strengthened by an earlier observation that the essential domain can be interchanged between the two enzymes. To examine possible functions of the P3 domain, four conserved nucleotides in the P3 domain of Saccharomyces cerevisiae RNase P RNA (RPR1) were randomized to create a library of all possible sequence combinations at those positions. Selection of functional genes in vivo identified permissible variations, and viable clones that caused yeast to exhibit conditional growth phenotypes were tested for defects in RNase P RNA and tRNA biosynthesis. Under nonpermissive conditions, the mutants had reduced maturation of the RPR1 RNA precursor, an expected phenotype in cases where RNase P holoenzyme assembly is defective. This loss of RPR1 RNA maturation coincided, as expected, with a loss of pre-tRNA maturation characteristic of RNase P defects. To test whether mutations at the conserved positions inhibited interactions with a particular protein, specific binding of the individual protein subunits to the RNA subunit was tested in yeast using the three-hybrid system. Pop1p, the largest subunit shared by RNases P and MRP, bound specifically to RPR1 RNA and the isolated P3 domain, and this binding was eliminated by mutations at the conserved P3 residues. These results indicate that Pop1p interacts with the P3 domain common to RNases P and MRP, and that this interaction is critical in the maturation of RNase P holoenzyme.     

Eukaryotic RNase P: role of RNA and protein subunits of a primordial catalytic ribonucleoprotein in RNA-based catalysis

Ribonuclease P (RNase P) is an essential enzyme that processes the 5' leader sequence of precursor tRNA. Eubacterial RNase P is an RNA enzyme, while its eukaryotic counterpart acts as catalytic ribonucleoprotein, consisting of RNA and numerous protein subunits. To study the latter form, we reconstitute human RNase P activity, demonstrating that the subunits H1 RNA, Rpp21, and Rpp29 are sufficient for 5' cleavage of precursor tRNA. The reconstituted RNase P precisely delineates its cleavage sites in various substrates and hydrolyzes the phosphodiester bond. Rpp21 and Rpp29 facilitate catalysis by H1 RNA, which seems to require a phylogenetically conserved pseudoknot structure for function. Unexpectedly, Rpp29 forms a catalytic complex with M1 RNA of E. coli RNase P. The results uncover the core components of eukaryotic RNase P, reveal its evolutionary origin in translation, and provide a paradigm for studying RNA-based catalysis by other nuclear and nucleolar ribonucleoprotein enzymes.     

Solution Structure of an Archaeal RNase P Binary Protein Complex: Formation of the 30-kDa Complex between Pyrococcus furiosus RPP21 and RPP29 Is Accompanied by Coupled Protein Folding and Highlights Critical Features for Protein-Protein and Protein-RNA Interactions

Ribonuclease P (RNase P) is a ribonucleoprotein (RNP) enzyme that catalyzes the Mg²⁺-dependent 5′ maturation of precursor tRNAs. In all domains of life, it is a ribozyme: the RNase P RNA (RPR) component has been demonstrated to be responsible for catalysis. However, the number of RNase P protein subunits (RPPs) varies from 1 in bacteria to 9 or 10 in eukarya. The archaeal RPR is associated with at least 4 RPPs, which function in pairs (RPP21-RPP29 and RPP30-POP5). We used solution NMR spectroscopy to determine the three-dimensional structure of the protein-protein complex comprising Pyrococcus furiosus RPP21 and RPP29. We found that the protein-protein interaction is characterized by coupled folding of secondary structural elements that participate in interface formation. In addition to detailing the intermolecular contacts that stabilize this 30-kDa binary complex, the structure identifies surfaces rich in conserved basic residues likely vital for recognition of the RPR and/or precursor tRNA. Furthermore, enzymatic footprinting experiments allowed us to localize the RPP21-RPP29 complex to the specificity domain of the RPR. These findings provide valuable new insights into mechanisms of RNP assembly and serve as important steps towards a three-dimensional model of this ancient RNP enzyme.     

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.     

Inhibition of bacterial RNase P by aminoglycoside-arginine conjugates

The potential of RNAs and RNA-protein (RNP) complexes as drug targets is currently being explored in various investigations. For example, a hexa-arginine derivative of neomycin (NeoR) and a tri-arginine derivative of gentamicin (R3G) were recently shown to disrupt essential RNP interactions between the trans-activator protein (Tat) and the Tat-responsive RNA (trans-activating region) in the human immunodeficiency virus (HIV) and also inhibit HIV replication in cell culture. Based on certain structural similarities, we postulated that NeoR and R3G might also be effective in disrupting RNP interactions and thereby inhibiting bacterial RNase P, an essential RNP complex involved in tRNA maturation. Our results indicate that indeed both NeoR and R3G inhibit RNase P activity from evolutionarily divergent pathogenic bacteria and do so more effectively than they inhibit partially purified human RNase P activity.     

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.     

Purification and characterization of Rpp25, an RNA-binding protein subunit of human ribonuclease P

In HeLa cells, ribonuclease P (RNase P), the tRNA processing enzyme consists of an RNA subunit (H1 RNA) associated with at least nine protein subunits, Rpp14, Rpp20, Rpp21, Rpp29 (hPop4), Rpp30, Rpp38, Rpp40, hPop1, and hPop5 (18.8 kDa). We report here the cloning and immuno-biochemical analysis of Rpp25, another protein subunit of RNase P. Polyclonal rabbit antibodies raised against recombinant Rpp25 recognize their corresponding antigens in RNase P-containing fractions purified from HeLa cells, and they also precipitate active holoenzyme. Furthermore, this protein has general RNA binding properties.     

Interactions between subunits of Saccharomyces cerevisiae RNase MRP support a conserved eukaryotic RNase P/MRP architecture

Ribonuclease MRP is an endonuclease, related to RNase P, which functions in eukaryotic pre-rRNA processing. In Saccharomyces cerevisiae, RNase MRP comprises an RNA subunit and ten proteins. To improve our understanding of subunit roles and enzyme architecture, we have examined protein-protein and protein–RNA interactions in vitro, complementing existing yeast two-hybrid data. In total, 31 direct protein–protein interactions were identified, each protein interacting with at least three others. Furthermore, seven proteins self-interact, four strongly, pointing to subunit multiplicity in the holoenzyme. Six protein subunits interact directly with MRP RNA and four with pre-rRNA. A comparative analysis with existing data for the yeast and human RNase P/MRP systems enables confident identification of Pop1p, Pop4p and Rpp1p as subunits that lie at the enzyme core, with probable addition of Pop5p and Pop3p. Rmp1p is confirmed as an integral subunit, presumably associating preferentially with RNase MRP, rather than RNase P, via interactions with Snm1p and MRP RNA. Snm1p and Rmp1p may act together to assist enzyme specificity, though roles in substrate binding are also indicated for Pop4p and Pop6p. The results provide further evidence of a conserved eukaryotic RNase P/MRP architecture and provide a strong basis for studies of enzyme assembly and subunit function.     

Genetic analysis of the structure and function of RNase P fromE. coli

A brief review of the genetic studies on ribonuclease P (RNase P) fromEscherichia coli is presented. Temperature-sensitive mutants ofE. coli defective in tRNA processing were isolated by screening cells which were unable to synthesize a suppressor tRNA at restrictive temperature. Structural analysis of accumulated tRNA precursors showed that the isolated mutants were defective in RNase P activity. Analyses of the mutants revealed that the enzyme is essential for the synthesis of all tRNA molecules in cells and that the enzymes consists of two subunits. Analyses of the isolated mutants revealed a possible domain structure of the RNA subunit of the enzyme.Abbreviations E. coli Escherichia coli - RNase P ribonuclease P     

Broadening the mission of an RNA enzyme

The “RNA World” hypothesis suggests that life developed from RNA enzymes termed ribozymes, which carry out reactions without assistance from proteins. Ribonuclease (RNase) P is one ribozyme that appears to have adapted these origins to modern cellular life by adding protein to the RNA core in order to broaden the potential functions. This RNA‐protein complex plays diverse roles in processing RNA, but its best‐understood reaction is pre‐tRNA maturation, resulting in mature 5' ends of tRNAs. The core catalytic activity resides in the RNA subunit of almost all RNase P enzymes but broader substrate tolerance is required for recognizing not only the diverse sequences of tRNAs, but also additional cellular RNA substrates. This broader substrate tolerance is provided by the addition of protein to the RNA core and allows RNase P to selectively recognize different RNAs, and possibly ribonucleoprotein (RNP) substrates. Thus, increased protein content correlated with evolution from bacteria to eukaryotes has further enhanced substrate potential enabling the enzyme to function in a complex cellular environment. J. Cell. Biochem. 108: 1244–1251, 2009. © 2009 Wiley‐Liss, Inc.     

Crystal Structure of Human Rpp20/Rpp25 Reveals Quaternary Level Adaptation of the Alba Scaffold as Structural Basis for Single-stranded RNA Binding

Ribonuclease P (RNase P) catalyzes the removal of 5′ leaders of tRNA precursors and its central catalytic RNA subunit is highly conserved across all domains of life. In eukaryotes, RNase P and RNase MRP, a closely related ribonucleoprotein enzyme, share several of the same protein subunits, contain a similar catalytic RNA core, and exhibit structural features that do not exist in their bacterial or archaeal counterparts. A unique feature of eukaryotic RNase P/MRP is the presence of two relatively long and unpaired internal loops within the P3 region of their RNA subunit bound by a heterodimeric protein complex, Rpp20/Rpp25. Here we present a crystal structure of the human Rpp20/Rpp25 heterodimer and we propose, using comparative structural analyses, that the evolutionary divergence of the single-stranded and helical nucleic acid binding specificities of eukaryotic Rpp20/Rpp25 and their related archaeal Alba chromatin protein dimers, respectively, originate primarily from quaternary level differences observed in their heterodimerization interface. Our work provides structural insights into how the archaeal Alba protein scaffold was adapted evolutionarily for incorporation into several functionally-independent eukaryotic ribonucleoprotein complexes.     

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.     

Structural organizations of yeast RNase P and RNase MRP holoenzymes as revealed by UV-crosslinking studies of RNA-protein interactions

Eukaryotic ribonuclease (RNase) P and RNase MRP are closely related ribonucleoprotein complexes involved in the metabolism of various RNA molecules including tRNA, rRNA, and some mRNAs. While evolutionarily related to bacterial RNase P, eukaryotic enzymes of the RNase P/MRP family are much more complex. Saccharomyces cerevisiae RNase P consists of a catalytic RNA component and nine essential proteins; yeast RNase MRP has an RNA component resembling that in RNase P and 10 essential proteins, most of which are shared with RNase P. The structural organizations of eukaryotic RNases P/MRP are not clear. Here we present the results of RNA-protein UV crosslinking studies performed on RNase P and RNase MRP holoenzymes isolated from yeast. The results indicate locations of specific protein-binding sites in the RNA components of RNase P and RNase MRP and shed light on the structural organizations of these large ribonucleoprotein complexes.     

RNase P from bacteria. Substrate recognition and function of the protein subunit

RNase P recognizes many different precursor tRNAs as well as other substrates and cleaves all of them accurately at the expected position. RNase P recognizes the tRNA structure of the precursor tRNA by a set of interactions between the catalytic RNA subunit and the T- and acceptor-stems mainly, although residues in the 5-leader sequence as well as the 3-terminal CCA are important. These conclusions have been reached by several studies on mutant precursor tRNAs as well as cross-linking studies between RNase P RNA and precursor tRNAs. The protein subunit of RNase P seems also to affect the way that the substrate is recognized as well as the range of substrates that can be used by RNase P, although the protein does not seem to interact directly with the substrates. The interaction between the protein and RNA subunits of RNase P has been extensively studiedin vitro. The protein subunit sequence is not highly conserved among bacteria, however different proteins are functionally equivalent as heterologous reconstitution of the RNase P holoenzyme can be achieved in many cases.Abbreviations C5 protein protein subunit fromE. coli RNase P - EGS external guide sequence - M1 RNA RNA subunit formE. coli RNase P - ptRNA precursor tRNA - RNase P ribonuclease P