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.     

Minor changes largely restore catalytic activity of archaeal RNase P RNA from Methanothermobacter thermoautotrophicus

The increased protein proportion of archaeal and eukaryal ribonuclease (RNase) P holoenzymes parallels a vast decrease in the catalytic activity of their RNA subunits (P RNAs) alone. We show that a few mutations toward the bacterial P RNA consensus substantially activate the catalytic (C-) domain of archaeal P RNA from Methanothermobacter, in the absence and presence of the bacterial RNase P protein. Large increases in ribozyme activity required the cooperative effect of at least two structural alterations. The P1 helix of P RNA from Methanothermobacter was found to be extended, which increases ribozyme activity (ca 200-fold) and stabilizes the tertiary structure. Activity increases of mutated archaeal C-domain variants were more pronounced in the context of chimeric P RNAs carrying the bacterial specificity (S-) domain of Escherichia coli instead of the archaeal S-domain. This could be explained by the loss of the archaeal S-domain''s capacity to support tight and productive substrate binding in the absence of protein cofactors. Our results demonstrate that the catalytic capacity of archaeal P RNAs is close to that of their bacterial counterparts, but is masked by minor changes in the C-domain and, particularly, by poor function of the archaeal S-domain in the absence of archaeal protein cofactors.     

In vitro selection of an archaeal RNase P RNA mimics natural variation

Archaeal and bacterial RNase P RNAs are similar in sequence and secondary structure, but in the absence of protein, the archaeal RNAs are much less active and require extreme ionic conditions for activity. To assess how readily the activity of the archaeal RNA alone could be improved by small changes in sequence, in vitro selection was used to generate variants of a Methanobacterium formicicum RNase P RNA: Bacillus subtilus pre-tRNA(Asp) self-cleaving conjugate RNA. Functional variants were generated with a spectrum of mutations that were predominately consistent with natural variation in this RNA. Variants generated from the selection had cleavage rates comparable to that of wild type; variants with improved cleavage rates or lower ionic requirements were not obtained. This suggests that the RNase P RNAs of Bacteria and Archaea are globally optimized and the basis for the large biochemical differences between these two types of RNase P RNA is distributed in the molecule.     

Solution structure and metal-ion binding of the P4 element from bacterial RNase P RNA

We determined the solution structure of two 27-nt RNA hairpins and their complexes with cobalt(III)-hexammine (Co(NH3)3+(6)) by NMR spectroscopy. The RNA hairpins used in this study are the P4 region from Escherichia coli RNase P RNA and a C-to-U mutant that confers altered divalent metal-ion specificity (Ca2+ replaces Mg2+) for catalytic activity of this ribozyme. Co(NH3)3+(6) is a useful spectroscopic probe for Mg(H2O)2+(6)-binding sites because both complexes have octahedral symmetry and have similar radii. The thermodynamics of binding to both RNA hairpins was studied using chemical shift changes upon titration with Mg2+, Ca2+, and Co(NH3)3+(6). We found that the equilibrium binding constants for each of the metal ions was essentially unchanged when the P4 model RNA hairpin was mutated, although the NMR structures show that the RNA hairpins adopt different conformations. In the C-to-U mutant a C.G base pair is replaced by U.G, and the conserved bulged uridine in the P4 wild-type stem shifts in the 3' direction by 1 nt. Intermolecular NOE cross-peaks between Co(NH3)3+(6) and RNA protons were used to locate the site of Co(NH3)3+(6) binding to both RNA hairpins. The metal ion binds in the major groove near a bulge loop, but is shifted 5' by more than 1 bp in the mutant. The change of the metal-ion binding site provides a possible explanation for changes in catalytic activity of the mutant RNase P in the presence of Ca2+.     

Phylogenetic comparative mutational analysis of the base-pairing between RNase P RNA and its substrate.

We have studied the base-pairing between the 3'-terminal CCA motif of a tRNA precursor and RNase P RNA by a phylogenetic mutational comparative approach. Thus, various derivatives of the Escherichia coli tRNA(Ser)Su1 precursor harboring all possible substitutions at either the first or the second C of the 3'-terminal CCA motif were generated. Cleavage site selection on these precursors was studied using mutant variants of M1 RNA, the catalytic subunit of E. coli RNase P, carrying changes at positions 292 or 293, which are involved in the interaction with the 3'-terminal CCA motif. From our data we conclude that these two C's in the substrate interact with the well-conserved G292 and G293 through canonical Watson-Crick base-pairing. Cleavage performed using reconstituted holoenzyme complexes suggests that this interaction also occurs in the presence of the C5 protein. Furthermore, we studied the interaction using various derivatives of RNase P RNAs from Mycoplasma hyopneumoniae and Mycobacterium tuberculosis. Our results suggest that the base-pairing between the 3'-terminal CCA motif and RNase P is present also in other bacterial RNase P-substrate complexes and is not limited to a particular bacterial species.     

Analysis of the tertiary structure of bacterial RNase P RNA

The ubiquitous occurrence of ribonuclease P (RNase P) as a ribonucleoprotein and the catalytic properties of bacterial RNase P RNAs indicate that RNA fulfills an ancient and important role in the function of this enzyme. This review focuses on efforts to determine the structure of the bacterial RNase P RNA ribozyme. Phylogenetic comparative analysis of a library of bacterial RNase P RNA sequences has resulted in a well-developed secondary structure model and allowed identification of some elements of tertiary structure. The native structure has been redesigned by circular permutation to facilitate intra- and inter-molecular crosslinking experiments in order to gain further structural information. The crosslinking constraints, together with the constraints provided by comparative analyses, have been incorporated into a first-order model of the structure of the ribozyme-substrate complex. The developing structural perspective allows the design of self-cleaving pre-tRNA-RNase P RNA conjugates which are useful tools for additional structure-probing experiments.Abbreviations cpRNA circularly permuted RNA     

Structure-function analysis in nuclear RNase P RNA

Eukaryotic ribonuclease P (RNase P) enzymes require both RNA and protein subunits for activityin vivo andin vitro. We have undertaken an analysis of the complex RNA subunit of the nuclear holoenzyme in an effort to understand its structure and its similarities to and differences from the bacterial ribozymes. Phylogenetic analysis, structure-sensitive RNA footprinting, and directed mutagenesis reveal conserved secondary and tertiary structures with both strong similarities to the bacterial consensus and distinctive features. The effects of mutations in the most highly conserved positions are being used to dissect the functions of individual subdomains.Abbreviations RPRI ribonucleaseP ribonucleoprotein 1 gene fromSaccharomyces cerevisiae - Pu purine ribonucleoside     

The contribution of peripheral stem-loops to the catalytic activity of archaeal RNase P RNA from Pyrococcus horikoshii OT3

We investigated the contribution of peripheral stem-loops to the catalytic activity of an archaeal RNase P RNA, PhopRNA, from Pyrococcus horikoshii OT3. PhopRNA mutants, in which the stem-loops were individually deleted, were prepared and characterized with respect to precursor tRNA (pre-tRNA) cleavage activity in the presence of five RNase P proteins. All the mutants retained the activity to some extent, indicating that they are moderately implicated in catalysis. Further characterization suggested that the stem-loops serve largely as binding sites for the proteins, and that their interactions are predominantly involved in stabilization of the active conformation of PhopRNA.     

Studies on Methanocaldococcus jannaschii RNase P reveal insights into the roles of RNA and protein cofactors in RNase P catalysis

Ribonuclease P (RNase P), a ribonucleoprotein (RNP) complex required for tRNA maturation, comprises one essential RNA (RPR) and protein subunits (RPPs) numbering one in bacteria, and at least four in archaea and nine in eukarya. While the bacterial RPR is catalytically active in vitro, only select euryarchaeal and eukaryal RPRs are weakly active despite secondary structure similarity and conservation of nucleotide identity in their putative catalytic core. Such a decreased archaeal/eukaryal RPR function might imply that their cognate RPPs provide the functional groups that make up the active site. However, substrate-binding defects might mask the ability of some of these RPRs, such as that from the archaeon Methanocaldococcus jannaschii (Mja), to catalyze precursor tRNA (ptRNA) processing. To test this hypothesis, we constructed a ptRNA-Mja RPR conjugate and found that indeed it self-cleaves efficiently (k(obs), 0.15 min(-1) at pH 5.5 and 55 degrees C). Moreover, one pair of Mja RPPs (POP5-RPP30) enhanced k(obs) for the RPR-catalyzed self-processing by approximately 100-fold while the other pair (RPP21-RPP29) had no effect; both binary RPP complexes significantly reduced the monovalent and divalent ionic requirement. Our results suggest a common RNA-mediated catalytic mechanism in all RNase P and help uncover parallels in RNase P catalysis hidden by plurality in its subunit make-up.     

RNase P RNA ofMycoplasma capricolum

There are at least six small stable RNAs inMycoplasma capricolum cells besides tRNAs and rRNAs. One of them, MCS5 RNA, is a homolog of RNase P RNA. The predicted secondary structure of this RNA is essentially the same as that of other eubacterial RNase P RNAs. MCS5 RNA is more similar to the RNase P RNA ofB. subtilis than to that ofE. coli. This is consistent with previous conclusions that mycoplasmas are phylogenetically related to the low G+C Gram-positive bacterial group. The major substrates for MCS5 RNA must be the precursors of tRNAs. The precursor of MCS6 RNA, which is a homolog of theE. coli 10Sa RNA, may also be a substrate for the MCS5 RNA because this RNA has a tRNA-like structure at its 5 and 3 ends.     

In search of RNase P RNA from microbial genomes

A simple procedure has been developed to quickly retrieve and validate the DNA sequence encoding the RNA subunit of ribonuclease P (RNase P RNA) from microbial genomes. RNase P RNA sequences were identified from 94% of bacterial and archaeal complete genomes where previously no RNase P RNA was annotated. A sequence was found in camelpox virus, highly conserved in all orthopoxviruses (including smallpox virus), which could fold into a putative RNase P RNA in terms of conserved primary features and secondary structure. New structure features of RNase P RNA that enable one to distinguish bacteria from archaea and eukarya were found. This RNA is yet another RNA that can be a molecular criterion to divide the living world into three domains (bacteria, archaea, and eukarya). The catalytic center of this RNA, and its detection from some environmental whole genome shotgun sequences, is also discussed.     

Crystal structure of archaeal RNase HII: a homologue of human major RNase H

BACKGROUND: RNases H are present in all organisms and cleave RNAs in RNA/DNA hybrids. There are two major types of RNases H that have little similarity in sequence, size and specificity. The structure of RNase HI, the smaller enzyme and most abundant in bacteria, has been extensively studied. However, no structural information is available for the larger RNase H, which is most abundant in eukaryotes and archaea. Mammalian RNase H participates in DNA replication, removal of the Okazaki fragments and possibly DNA repair. RESULTS: The crystal structure of RNase HII from the hypothermophile Methanococcus jannaschii, which is homologous to mammalian RNase H, was solved using a multiwavelength anomalous dispersion (MAD) phasing method at 2 A resolution. The structure contains two compact domains. Despite the absence of sequence similarity, the large N-terminal domain shares a similar fold with the RNase HI of bacteria. The active site of RNase HII contains three aspartates: Asp7, Asp112 and Asp149. The nucleotide-binding site is located in the cleft between the N-terminal and C-terminal domains. CONCLUSIONS: Despite a lack of any detectable similarity in primary structure, RNase HII shares a similar structural domain with RNase HI, suggesting that the two classes of RNases H have a common catalytic mechanism and possibly a common evolutionary origin. The involvement of the unique C-terminal domain in substrate recognition explains the different reaction specificity observed between the two classes of RNase H.     

Phylogenetic analysis of the structure of RNase MRP RNA in yeasts

RNase MRP is a ribonucleoprotein enzyme involved in processing precursor rRNA in eukaryotes. To facilitate our structure-function analysis of RNase MRP from Saccharomyces cerevisiae, we have determined the likely secondary structure of the RNA component by a phylogenetic approach in which we sequenced all or part of the RNase MRP RNAs from 17 additional species of the Saccharomycetaceae family. The structure deduced from these sequences contains the helices previously suggested to be common to the RNA subunit of RNase MRP and the related RNA subunit of RNase P, an enzyme cleaving tRNA precursors. However, outside this common region, the structure of RNase MRP RNA determined here differs from a previously proposed universal structure for RNase MRPs. Chemical and enzymatic structure probing analyses were consistent with our revised secondary structure. Comparison of all known RNase MRP RNA sequences revealed three regions with highly conserved nucleotides. Two of these regions are part of a helix implicated in RNA catalysis in RNase P, suggesting that RNase MRP may cleave rRNA using a similar catalytic mechanism.     

Protein cofactors and substrate influence Mg2+-dependent structural changes in the catalytic RNA of archaeal RNase P

The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg²⁺-dependent cleavage of the 5′ leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ∼250–500 mM Mg²⁺ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg²⁺ requirement to ∼10–20 mM and improves the rate and fidelity of cleavage. To understand the Mg²⁺- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPR_{C domain} and Cy5-RPR_{S domain}), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg²⁺-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg²⁺ cooperativity. Our findings highlight how Mg²⁺ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.     

Cooperative RNP assembly: complementary rescue of structural defects by protein and RNA subunits of archaeal RNase P

Ribonuclease P (RNase P) is a ribonucleoprotein complex that utilizes a Mg(2+)-dependent RNA catalyst to cleave the 5' leader of precursor tRNAs (pre-tRNAs) and generate mature tRNAs. The bacterial RNase P protein (RPP) aids RNase P RNA (RPR) catalysis by promoting substrate binding, Mg(2+) coordination and product release. Archaeal RNase P comprises an RPR and at least four RPPs, which have eukaryal homologs and function as two binary complexes (POP5·RPP30 and RPP21·RPP29). Here, we employed a previously characterized substrate-enzyme conjugate [pre-tRNA(Tyr)-Methanocaldococcus jannaschii (Mja) RPR] to investigate the functional role of a universally conserved uridine in a bulge-helix structure in archaeal RPRs. Deletion of this bulged uridine resulted in an 80-fold decrease in the self-cleavage rate of pre-tRNA(Tyr)-MjaΔU RPR compared to the wild type, and this defect was partially ameliorated upon addition of either RPP pair. The catalytic defect in the archaeal mutant RPR mirrors that reported in a bacterial RPR and highlights a parallel in their active sites. Furthermore, an N-terminal deletion mutant of Pyrococcus furiosus (Pfu) RPP29 that is defective in assembling with its binary partner RPP21, as assessed by isothermal titration calorimetry and NMR spectroscopy, is functional when reconstituted with the cognate Pfu RPR. Collectively, these results indicate that archaeal RPPs are able to compensate for structural defects in their cognate RPR and vice-versa, and provide striking examples of the cooperative subunit interactions critical for driving archaeal RNase P toward its functional conformation.