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 RNase P associated with HeLa cell mitochondria contains an essential RNA component identical in sequence to that of the nuclear RNase P

The mitochondrion-associated RNase P activity (mtRNase P) was extensively purified from HeLa cells and shown to reside in particles with a sedimentation constant ( approximately 17S) very similar to that of the nuclear enzyme (nuRNase P). Furthermore, mtRNase P, like nuRNase P, was found to process a mitochondrial tRNA(Ser(UCN)) precursor [ptRNA(Ser(UCN))] at the correct site. Treatment with micrococcal nuclease of highly purified mtRNase P confirmed earlier observations indicating the presence of an essential RNA component. Furthermore, electrophoretic analysis of 3'-end-labeled nucleic acids extracted from the peak of glycerol gradient-fractionated mtRNase P revealed the presence of a 340-nucleotide RNA component, and the full-length cDNA of this RNA was found to be identical in sequence to the H1 RNA of nuRNase P. The proportions of the cellular H1 RNA recovered in the mitochondrial fractions from HeLa cells purified by different treatments were quantified by Northern blots, corrected on the basis of the yield in the same fractions of four mitochondrial nucleic acid markers, and shown to be 2 orders of magnitude higher than the proportions of contaminating nuclear U2 and U3 RNAs. In particular, these experiments revealed that a small fraction of the cell H1 RNA (of the order of 0.1 to 0.5%), calculated to correspond to approximately 33 to approximately 175 intact molecules per cell, is intrinsically associated with mitochondria and can be removed only by treatments which destroy the integrity of the organelles. In the same experiments, the use of a probe specific for the RNA component of RNase MRP showed the presence in mitochondria of 6 to 15 molecules of this RNA per cell. The available evidence indicates that the levels of mtRNase P detected in HeLa cells should be fully adequate to satisfy the mitochondrial tRNA synthesis requirements of these cells.     

Cleavage mediated by the P15 domain of bacterial RNase P RNA

Independently folded domains in RNAs frequently adopt identical tertiary structures regardless of whether they are in isolation or are part of larger RNA molecules. This is exemplified by the P15 domain in the RNA subunit (RPR) of the universally conserved endoribonuclease P, which is involved in the processing of tRNA precursors. One of its domains, encompassing the P15 loop, binds to the 3'-end of tRNA precursors resulting in the formation of the RCCA-RNase P RNA interaction (interacting residues underlined) in the bacterial RPR-substrate complex. The function of this interaction was hypothesized to anchor the substrate, expose the cleavage site and result in re-coordination of Mg(2+) at the cleavage site. Here we show that small model-RNA molecules (~30 nt) carrying the P15-loop mediated cleavage at the canonical RNase P cleavage site with significantly reduced rates compared to cleavage with full-size RPR. These data provide further experimental evidence for our model that the P15 domain contributes to both substrate binding and catalysis. Our data raises intriguing evolutionary possibilities for 'RNA-mediated' cleavage of RNA.     

Binding and cleavage of unstructured RNA by nuclear RNase P

Ribonuclease P (RNase P) is an essential endoribonuclease for which the best-characterized function is processing the 5' leader of pre-tRNAs. Compared to bacterial RNase P, which contains a single small protein subunit and a large catalytic RNA subunit, eukaryotic nuclear RNase P is more complex, containing nine proteins and an RNA subunit in Saccharomyces cerevisiae. Consistent with this, nuclear RNase P has been shown to possess unique RNA binding capabilities. To understand the unique molecular recognition of nuclear RNase P, the interaction of S. cerevisiae RNase P with single-stranded RNA was characterized. Unstructured, single-stranded RNA inhibits RNase P in a size-dependent manner, suggesting that multiple interactions are required for high affinity binding. Mixed-sequence RNAs from protein-coding regions also bind strongly to the RNase P holoenzyme. However, in contrast to poly(U) homopolymer RNA that is not cleaved, a variety of mixed-sequence RNAs have multiple preferential cleavage sites that do not correspond to identifiable consensus structures or sequences. In addition, pre-tRNA(Tyr), poly(U)(50) RNA, and mixed-sequence RNA cross-link with purified RNase P in the RNA subunit Rpr1 near the active site in "Conserved Region I," although the exact positions vary. Additional contacts between poly(U)(50) and the RNase P proteins Rpr2p and Pop4p were identified. We conclude that unstructured RNAs interact with multiple protein and RNA contacts near the RNase P RNA active site, but that cleavage depends on the nature of interaction with the active site.     

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 P7Δ 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 Mg²⁺. The results suggest that with at least one important exception of specific binding, these sites mainly map areas of diffuse association of magnesium ions.     

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.     

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.     

The precursor tRNA 3'-CCA interaction with Escherichia coli RNase P RNA is essential for catalysis by RNase P in vivo

The L15 region of Escherichia coli RNase P RNA forms two Watson-Crick base pairs with precursor tRNA 3'-CCA termini (G292-C75 and G293-C74). Here, we analyzed the phenotypes associated with disruption of the G292-C75 or G293-C74 pair in vivo. Mutant RNase P RNA alleles (rnpBC292 and rnpBC293) caused severe growth defects in the E. coli rnpB mutant strain DW2 and abolished growth in the newly constructed mutant strain BW, in which chromosomal rnpB expression strictly depended on the presence of arabinose. An isosteric C293-G74 base pair, but not a C292-G75 pair, fully restored catalytic performance in vivo, as shown for processing of precursor 4.5S RNA. This demonstrates that the base identity of G292, but not G293, contributes to the catalytic process in vivo. Activity assays with mutant RNase P holoenzymes assembled in vivo or in vitro revealed that the C292/293 mutations cause a severe functional defect at low Mg2+ concentrations (2 mM), which we infer to be on the level of catalytically important Mg2+ recruitment. At 4.5 mM Mg2+, activity of mutant relative to the wild-type holoenzyme, was decreased only about twofold, but 13- to 24-fold at 2 mM Mg2+. Moreover, our findings make it unlikely that the C292/293 phenotypes include significant contributions from defects in protein binding, substrate affinity, or RNA degradation. However, native PAGE experiments revealed nonidentical RNA folding equilibria for the wild-type versus mutant RNase P RNAs, in a buffer- and preincubation-dependent manner. Thus, we cannot exclude that altered folding of the mutant RNAs may have also contributed to their in vivo defect.     

Enzymatic RNA synthesis and RNase P

TransferRNA recognition was used as leit-motiv in the illustration of possible links between a hypothetical primordial RNA world and the contemporary DNA world. In an RNA world, proto-tRNA could have functioned as replication origin and as primitive telomere. Possibly, this primitive structure is preserved in a universal substrate for modern tRNA-specific enzymes. The combination of acceptor stem and T arm (plus a linker) was finally revealed as sufficient for the recognition by prokaryotic and eukaryotic RNase P, as well as other tRNA enzymes. In modern life forms, a tRNA-like element in viral RNAs still serves as replication origin, and furthermore, the recognition of similar structures as cryptic promoters is universally conserved for template-dependent RNA polymerases. Another common property of modern polymerases is their high, but clearly limited and condition-dependent substrate specificity. Very likely, also substrate recognition by primitive polymerases was not more stringent, and this lead to the ocurrence of mixed nucleic acids as intermediates in the transition from genomic RNA to contemporary genomic DNA.Abbreviations (d)NTP (deoxy)nucleoside triphosphate - nt nucleotide(s)     

Modular construction of a tertiary RNA structure: the specificity domain of the Bacillus subtilis RNase P RNA

The structure of the specificity domain (S-domain) of the Bacillus subtilis RNase P RNA has been proposed to be composed of a core and a buttress module, analogous to the bipartite structure of the P4-P6 domain of the Tetrahymena group I ribozyme. The core module is the functional unit of the S-domain and contains the binding site for the T stem-loop of a tRNA. The buttress module provides structural stability to the core module and consists of a GA3 tetraloop and its receptor. To explicitly test the hypothesis that modular construction can describe the structure of the S-domain and is a useful RNA design strategy, we analyzed the equilibrium folding and substrate binding of three classes of S-domain mutants. Addition or deletion of a base pair in the helical linker region between the modules only modestly destabilizes the tertiary structure. tRNA binding selectivity is affected in one but not in two other mutants of this class. Elimination of the GA3 tetraloop-receptor interactions significantly destabilizes the core module and results in the loss of tRNA binding selectivity. Replacing the buttress module with that of a homologous RNase P RNA maintains the tRNA binding selectivity. Overall, we have observed that the linker regions between the two modules can tolerate moderate structural changes and that the buttress modules can be shuffled between homologous S-domains. These results suggest that it is feasible to design an RNA using a buttress module to stabilize a functional module.     

A conserved RNA motif involved in divalent cation utilization by nuclear RNase P.

Catalytic RNAs are metalloenzymes that require precise coordination of divalent cation cofactors. In RNase P RNA, a conserved structural subdomain that has been implicated in magnesium coordination contains the consensus sequence acAGaRA. Randomization mutagenesis of the analogous sequence in the Saccharomyces cerevisiae nuclear RNase P RNA gene, RPR1, gave viable sequence variants that confer magnesium-correctable growth defects and are defective in magnesium cofactor utilization by the RNase P holoenzyme in vitro. Kinetic analysis of the defective holoenzymes suggests that the primary effects were on catalytic rate, rather than substrate recognition. The possible involvement of this RNA subdomain in catalysis is discussed.     

Archaeal RNase P has multiple protein subunits homologous to eukaryotic nuclear RNase P proteins

Although archaeal RNase P RNAs are similar in both sequence and structure to those of Bacteria rather than eukaryotes, and heterologous reconstitution between the Bacillus subtilis RNase P protein and some archaeal RNase P RNAs has been demonstrated, no archaeal protein sequences with similarity to any known bacterial RNase P protein subunit have been identified, and the density of Methanothermobacter thermoautotrophicus RNase P in Cs2SO4 (1.42 g/mL) is inconsistent with a single small bacterial-like protein subunit. Four hypothetical open reading frames (MTH11, MTH687, MTH688, and MTH1618) were identified in the genome of M. thermoautotrophicus that have sequence similarity to four of the nine Saccharomyces cerevisiae RNase P protein subunits: Pop4p, Pop5p, Rpp1p, and Rpr2p, respectively. Polyclonal antisera generated to recombinant Mth11p, Mth687p, Mth688p, and Mth1618p each recognized a protein of the predicted molecular weight in western blots of partially purified M. thermoautotrophicus RNase P, and immunoprecipitated RNase P activity from the same partially purified preparation. RNase P in Archaea is therefore composed of an RNA subunit similar to bacterial RNase P RNA and multiple protein subunits similar to those in the eukaryotic nucleus.     

Slow folding kinetics of RNase P RNA.

Understanding the folding mechanisms of large, highly structured RNAs is important for understanding how these molecules carry out their function. Although models for the three-dimensional architecture of several large RNAs have been constructed, the process by which these structures are formed is only now beginning to be explored. The kinetic folding pathway of the Tetrahymena ribozyme involves multiple intermediates and both Mg2+-dependent and Mg2+-independent steps. To determine whether this general mechanism is representative of folding of other large RNAs, a study of RNase P RNA folding was undertaken. We show, using a kinetic oligonucleotide hybridization assay, that there is at least one slow step on the folding pathway of RNase P RNA, resulting in conformational changes in the P7 helix region on the minute timescale. Although this folding event requires the presence of Mg2+, the slow step itself does not involve Mg2+ binding. The P7 and P2 helix regions exhibit distinctly different folding behavior and ion dependence, implying that RNase P folding is likely to be a complex process. Furthermore, there are distinct similarities in the folding of RNase P RNA from both Bacillus subtilis and Escherichia coli, indicating that the folding pathway may also be conserved along with the final structure. The slow folding kinetics, Mg2+-independence of the rate, and existence of intermediates are basic features of the folding mechanism of the Tetrahymena group I intron that are also found in RNase P RNA, suggesting these may be general features of the folding of large RNAs.     

The P15-loop of Escherichia coli RNase P RNA is an autonomous divalent metal ion binding domain.

We have studied the structure and divalent metal ion binding of a domain of the ribozyme RNase P RNA that is involved in base pairing with its substrate. Our data suggest that the folding of this internal loop, the P15-loop, is similar irrespective of whether it is part of the full-length ribozyme or part of a model RNA molecule. We also conclude that this element constitutes an autonomous divalent metal ion binding domain of RNase P RNA and our data suggest that certain specific chemical groups within the P15-loop participate in coordination of divalent metal ions. Substitutions of the Sp- and Rp-oxygens with sulfur at a specific position in this loop result in a 2.5-5-fold less active ribozyme, suggesting that Mg2+ binding at this position contributes to function. Our findings strengthen the concept that small RNA building blocks remain basically unchanged when removed from their structural context and thus can be used as models for studies of their potential function and structure within native RNA molecules.