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Query: EC:3.1.26.5 (RNase P)
 1,348 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

RNase P preparations from Escherichia coli can be separated into RNA and protein by chromatography, in buffers containing 7 M urea, on Sephadex G-200, DEAE-Sephadex, or CM-Sephadex columns. Neither RNA nor protein components alone exhibits any RNase activity. RNase P activity can be reconstituted by mixing separated RNA and protein components in buffer containing 7M urea followed by dialysis of this mixture to remove the urea. Of several purified RNAs tried, only M2 RNA, the RNA species found in purified RNase P, is active in the reconstitution experiments.
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PMID:Reconstitution of RNase P activity from inactive RNA and protein. 38 49

The folding thermodynamics and kinetics for the ribozyme from Bacillus subtilis RNase P are analyzed using circular dichroism and UV absorbance spectroscopies and catalytic activity. At 37 degrees C, the addition of Mg2+ (Kd approximately 50 microM) to the unfolded state produces an intermediate state within 1 ms which contains a comparable amount of secondary structure as the native ribozyme. The subsequent transition to the native state (Kd[Mg] approximately 0.8 mM, Hill coefficient approximately 3.5) has a half-life of hundreds of seconds as measured by circular dichroism at 278 nm and by a ribozyme activity assay. Surprisingly, the formation of the native structure is accelerated strongly by the addition of a denaturant; approximately 30-fold at 4.5 M urea. Thus, the rate-limiting step entails the disruption of a considerable number of interactions. The folding of this, and presumably other large RNAs, is slow due to the structural rearrangement of kinetically trapped species. Taken together with previous submillisecond relaxation kinetics of tRNA tertiary structure, we suggest that error-free RNA folding can be on the order of milliseconds.
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PMID:Intermediates and kinetic traps in the folding of a large ribozyme revealed by circular dichroism and UV absorbance spectroscopies and catalytic activity. 936 Jun 10

Ribonuclease P is a ribonucleoprotein complex that catalyzes the essential 5' maturation of all precursor tRNA molecules. The protein component both alters the conformation of the RNA component and enhances the substrate affinity and specificity. To facilitate biochemical and biophysical studies, the protein component of Bacillus subtilis ribonuclease P (RNase P) was overproduced in Escherichia coli using the native amino acid sequence with the initial 20 codons optimized for expression in E.coli . A simple purification procedure using consecutive cation exchange chromatography steps in the presence and absence of urea was developed to purify large quantities of P protein without contaminating nucleic acids. The identity of the recombinant protein as a cofactor of RNase P was established by its ability to stimulate the activity of the RNA component in low ionic strength buffer in a 1:1 stoichiometry. Circular dichroism studies indicate that P protein is a combination of alpha-helix and beta-sheet secondary structures and is quite stable, with a T m of 67 degrees C. The described methods facilitated the large scale purification of homogeneous, RNA-free P protein required for high resolution crystallographic analyses and may be useful for the preparation of other RNA binding proteins.
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PMID:Expression, purification and characterization of the recombinant ribonuclease P protein component from Bacillus subtilis. 962 4

The folding thermodynamics of the catalytic domain from the Bacillus subtilis RNase P RNA is analyzed using circular dichroism and fluorescence spectroscopies, hydroxyl radical protection, and catalytic activity. Folding of this 255-nucleotide ribozyme can be described with three populated species: unfolded (U), intermediate (I), and native (N) states. The U-to-I transition primarily involves secondary structure formation, whereas the I-to-N transition is dominated by tertiary structure formation. The I-to-N transition is highly cooperative as indicated by the coincidence of the four probes applied here. Two isothermal methods are used to determine the stability of the N state relative to the I state at 10 and 37 degrees C. The first method measures the extent of Mg(2+)-induced folding without urea or at constant urea concentrations. The second method measures the extent of urea-induced unfolding at constant Mg(2+) concentrations. Via application of a cooperative binding analysis, the Mg(2+) transition midpoint (K(Mg)), the Hill constant (n), and the urea-dependent surface burial parameter (m value) determined by both methods are identical, indicating that they report the same, reversible folding event. Three conclusions can be drawn from these results. (i) The folding free energy of a Mg(2+)-dependent tertiary RNA structure can be described by the K(Mg) and n parameters according to a cooperative Mg(2+) binding model. (ii) The Hill constant for this tertiary RNA structure probably represents the differential number of Mg(2+) ions bound in the I-to-N transition. (iii) Under physiological conditions, the stability of this large ribozyme is similar to that of small globular proteins.
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PMID:A thermodynamic framework and cooperativity in the tertiary folding of a Mg2+-dependent ribozyme. 1060 17

We apply synchrotron-based small-angle X-ray scattering to investigate the relationship between compaction, metal binding, and structure formation of two RNAs at 37 degrees C: the 76 nucleotide yeast tRNA(Phe) and the 255 nucleotide catalytic domain of the Bacillus subtilis RNase P RNA. For both RNAs, this method provides direct evidence for the population of a distinct folding intermediate. The relative compaction between the intermediate and the native state does not correlate with the size of the RNA but does correlate well with the amount of surface burial as quantified previously by the urea-dependent m-value. The total compaction process can be described in two major stages. Starting from a completely unfolded state (4-8 M urea, no Mg(2+)), the major amount of compaction occurs upon the dilution of the denaturant and the addition of micromolar amounts of Mg(2+) to form the intermediate. The native state forms in a single transition from the intermediate state upon cooperative binding of three to four Mg(2+) ions. The characterization of this intermediate by small-angle X-ray scattering lends strong support for the cooperative Mg(2+)-binding model to describe the stability of a tertiary RNA.
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PMID:Mg2+-dependent compaction and folding of yeast tRNAPhe and the catalytic domain of the B. subtilis RNase P RNA determined by small-angle X-ray scattering. 1099 49

Despite a growing literature on the folding of RNA, our understanding of tertiary folding in large RNAs derives from studies on a small set of molecular examples, with primary focus on group I introns and RNase P RNA. To broaden the scope of RNA folding models and to better understand group II intron function, we have examined the tertiary folding of a ribozyme (D135) that is derived from the self-splicing ai5gamma intron from yeast mitochondria. The D135 ribozyme folds homogeneously and cooperatively into a compact, well-defined tertiary structure that includes all regions critical for active-site organization and substrate recognition. When D135 was treated with increasing concentrations of Mg(2+) and then subjected to hydroxyl radical footprinting, similar Mg(2+) dependencies were seen for internalization of all regions of the molecule, suggesting a highly cooperative folding behavior. In this work, we show that global folding and compaction of the molecule have the same magnesium dependence as the local folding previously observed. Furthermore, urea denaturation studies indicate highly cooperative unfolding of the ribozyme that is governed by thermodynamic parameters similar to those for forward folding. In fact, D135 folds homogeneously and cooperatively from the unfolded state to its native, active structure, thereby demonstrating functional reversibility in RNA folding. Taken together, the data are consistent with two-state folding of the D135 ribozyme, which is surprising given the size and multi-domain structure of the RNA. The findings establish that the accumulation of stable intermediates prior to formation of the native state is not a universal feature of RNA folding and that there is an alternative paradigm in which the folding landscape is relatively smooth, lacking rugged features that obstruct folding to the native state.
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PMID:An alternative route for the folding of large RNAs: apparent two-state folding by a group II intron ribozyme. 1463 93

Understanding the interconversion between thermodynamically distinguishable states present in a protein folding pathway provides not only the kinetics and energetics of protein folding but also insights into the functional roles of these states in biological systems. The protein component of the bacterial RNase P holoenzyme from Bacillus subtilis (P protein) was previously shown to be unfolded in the absence of its cognate RNA or other anionic ligands. P protein was used in this study as a model system to explore general features of intrinsically disordered protein (IDP) folding mechanisms. The use of trimethylamine N-oxide (TMAO), an osmolyte that stabilizes the unliganded folded form of the protein, enabled us to study the folding process of P protein in the absence of ligand. Transient stopped-flow kinetic traces at various final TMAO concentrations exhibited multiphasic kinetics. Equilibrium "cotitration" experiments were performed using both TMAO and urea during the titration to produce a urea-TMAO titration surface of P protein. Both kinetic and equilibrium studies show evidence of a previously undetected intermediate state in the P protein folding process. The intermediate state is significantly populated, and the folding rate constants are relatively slow compared to those of intrinsically folded proteins similar in size and topology. The experiments and analysis described serve as a useful example for mechanistic folding studies of other IDPs.
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PMID:Osmolyte-induced folding of an intrinsically disordered protein: folding mechanism in the absence of ligand. 2047 78