Comparative structure analysis of vertebrate ribonuclease P RNA.

Ribonuclease P cleaves 5'-precursor sequences from pre-tRNAs. All cellular RNase P holoenzymes contain homologous RNA elements; the eucaryal RNase P RNA, in contrast to the bacterial RNA, is catalytically inactive in the absence of the protein component(s). To understand the function of eucaryal RNase P RNA, knowledge of its structure is needed. Considerable effort has been devoted to comparative studies of the structure of this RNA from diverse organisms, including eucaryotes, primarily fungi, but also a limited set of vertebrates. The substantial differences in the sequences and structures of the vertebrate RNAs from those of other organisms have made it difficult to align the vertebrate sequences, thus limiting comparative studies. To expand our understanding of the structure of diverse RNase P RNAs, we have isolated by PCR and sequenced 13 partial RNase P RNA genes from 11 additional vertebrate taxa representing most extant major vertebrate lineages. Based on a recently proposed structure of the core elements of RNase P RNA, we aligned the sequences and propose a minimum consensus secondary structure for the vertebrate RNase P RNA.     

Manganese(II) as a paramagnetic probe of the tertiary structure of transfer RNA.

The effect of manganese on both the low field (10--15 ppm) and the high field (o--3 ppm) NMR spectra of unfractionated tRNA and yeast tRNAPhe has been investigated. Trace amounts of Mn2+ cause selective broadening of resonances which are assigned to specific tertiary interactions. The order in which resonances broaden is the same as the order in which they are stabilized by the addition of magnesium, namely s4U8 - A14, U33 and A58 - T54. From this we conclude that three of the strong binding sites probably are the same for both Mn2+ and Mg2+, and that these sites are located close to the tertiary interactions which are stabilized by the strongly bound metals. The broadening data, taken in conjunction with published X-ray data on yeast tRNAPhe, permit us to suggest some plausible locations for the strong binding sites.     

Comparative analysis of ribonuclease P RNA structure in Archaea.

Although the structure of the catalytic RNA component of ribonuclease P has been well characterized in Bacteria, it has been little studied in other organisms, such as the Archaea. We have determined the sequences encoding RNase P RNA in eight euryarchaeal species: Halococcus morrhuae, Natronobacterium gregoryi, Halobacterium cutirubrum, Halobacteriurn trapanicum, Methanobacterium thermoautotrophicum strains deltaH and Marburg, Methanothermus fervidus and Thermococcus celer strain AL-1. On the basis of these and previously available sequences from Sulfolobus acidocaldarius, Haloferax volcanii and Methanosarcina barkeri the secondary structure of RNase P RNA in Archaea has been analyzed by phylogenetic comparative analysis. The archaeal RNAs are similar in both primary and secondary structure to bacterial RNase P RNAs, but unlike their bacterial counterparts these archaeal RNase P RNAs are not by themselves catalytically proficient in vitro.     

Deoxyribonuclease II as a probe for chromatin structure. I. Location of cleavage sites

DNAase II has been shown to cleave condensed mouse liver chromatin at 100-bp² intervals while chromatin in the extended form is cleaved at 200-bp intervals (Altenburger et al., 1976). Evidence is presented here that DNA digestion patterns of a half-nucleosomal periodicity are also obtained upon DNAase II digestion of chicken erythrocyte nuclei and yeast nuclei, both of which differ in their repeat lengths (210 and 165 bp) from mouse liver chromatin. In the digestion of mouse liver nuclei a shift from the 100-bp to the 200-bp cleavage mode takes place when the concentration of monovalent cations present during digestion is decreased below 1 mM. When soluble chromatin prepared by micrococcal nuclease is digested with DNAase II the same type of shift occurs, albeit at higher ionic strength.In order to map the positions of the DNAase II cleavage sites on the DNA relative to the positions of the nucleosome cores, the susceptibility of DNAase II-derived DNA termini to exonuclease III was investigated. In addition, oligonucleosome fractions from HaeIII and micrococcal nuclease digests were end-labelled with polynucleotide kinase and digested with DNAase II under conditions leading to 100 and 200-bp digestion patterns. Analysis of the chain lengths of the resulting radioactively labelled fragments together with the results of the exonuclease assay allow the following conclusions. In the 200-bp digestion mode, DNAase II cleaves exclusively in the internucleosomal linker region. Also in the 100-bp mode cleavage occurs initially in the linker region. Subsequently, DNAase II cleaves at intranucleosomal locations, which are not, however, in the centre of the nucleosome but instead around positions 20 and 125 of the DNA associated with the nucleosome core. At late stages of digestion intranucleosomal cuts predominate and linkers that are still intact are largely resistant to DNAase II due to interactions between adjacent nucleosomes. These findings offer an explanation for the sensitivity of DNAase II to the higher-order structure of chromatin.     

Phenanthroline-Cu(II) cleavage as a probe of rRNA structure

Chemical cleavage is developing into a powerful tool for analysis and characterization of nucleic acids. Phenanthroline-Cu(II) cleavage has been used extensively for studies of DNA for the last two decades, but recently has been applied to structural studies of RNA as well. This approach has been used to study the structure and structural changes occurring in ribosomal RNA within the ribosomes. In this article we discuss the mechanism by which phenanthroline cleaves, the applications possible using this approach, and the results that can be obtained. Protocols for use of phenanthroline are outlined as well.     

Pyrrolo-C as a fluorescent probe for monitoring RNA secondary structure formation

Pyrrolo-C (PC), or 3-[beta-D-2-ribofuranosyl]-6-methylpyrrolo[2,3-d]pyrimidin-2(3H)-one, is a fluorescent analog of the nucleoside cytidine that retains its Watson-Crick base-pairing capacity with G. Due to its red-shifted absorbance, it can be selectively excited in the presence of natural nucleosides, making it a potential site-specific probe for RNA structure and dynamics. Similar to 2-aminopurine nucleoside, which base-pairs with uridine (or thymidine), PC's fluorescence becomes reversibly quenched upon base-pairing, most likely due to stacking interactions with neighboring bases. To test its utility as an RNA probe, we examined PC's fluorescent properties over a wide range of ionic strengths, pH, organic cosolvents, and temperatures. Incorporation of PC into a single-stranded RNA results in an approximately 60% reduction of fluorescence intensity, while duplex formation reduces the fluorescence by approximately 75% relative to the free ribonucleoside. We find that the fluorescence intensity of PC is only moderately affected by ionic strength, pH, and temperature, while it is slightly enhanced by organic cosolvents, making it a versatile probe for a broad range of buffer conditions. We demonstrate two applications for PC: fluorescent measurements of the kinetics of formation and dissociation of an RNA/DNA complex, and fluorescent monitoring of the thermal denaturation of the central segment of an RNA duplex. Taken together, our data showcase the potential of pyrrolo-C as an effective fluorescent probe to study RNA structure, dynamics, and function, complementary to the popular 2-aminopurine ribonucleoside.     

Structure and evolution of ribonuclease P RNA.

Eubacterial RNase P contains a catalytic RNA that cleaves 5' leader sequences from precursor tRNAs. We review the current understanding of RNase P RNA structure and evolution, from the perspective of phylogenetic comparative analysis.     

Use of terbium as a probe of tRNA tertiary structure and folding

Lanthanide metals such as terbium have previously been shown to be useful for mapping metal-binding sites in RNA. Terbium binds to the same sites on RNA as magnesium, however, with a much higher affinity. Thus, low concentrations of terbium ions can easily displace magnesium and promote phosphodiester backbone scission. At higher concentrations, terbium cleaves RNA in a sequence-independent manner, with a preference for single-stranded, non-Watson-Crick base-paired regions. Here, we show that terbium is a sensitive probe of human tRNALys,3 tertiary structure and folding. When 1 microM tRNA is used, the optimal terbium ion concentration for detecting Mg2+-induced tertiary structural changes is 50-60 microM. Using these concentrations of RNA and terbium, a magnesium-dependent folding transition with a midpoint (KMg) of 2.6 mM is observed for unmodified human tRNALys,3. At lower Tb3+ concentrations, cleavage is restricted to nucleotides that constitute specific metal-binding pockets. This small chemical probe should also be useful for detecting protein induced structural changes in RNA.     

Recent approaches to probe functional groups in ribonuclease P RNA by modification interference

Modification interference is a powerful method to identify important functional groups in RNA molecules. We review here recent developments of techniques to screen for chemical modifications that interfere with (i) binding of(pre-)tRNA to bacterial RNase P RNA or (ii) pre-tRNA cleavage by this ribozyme. For example, two studies have analyzed positions at which a substitution of sulfur for thepro-Rp oxygen affects tRNA binding [1] or catalysis [2]. The results emphasize the functional key role of a central core element present in all known RNase P RNA subunits. The four sulfur substitutions identified in one study [2] to inhibit the catalytic step also interfered with binding of tRNA toE. coli RNase P RNA [1]. This suggests that losses in binding energy due to the modification at these positions affect the enzyme-substrate and the enzyme-transition state complex. In addition, the two studies have revealed, for the first time, sites of direct metal ion coordination in RNase P RNA. The potentials, limitations and interpretational ambiguities of modification interference experiments as well as factors influencing their outcome are discussed.Abbreviations nt nucleotide(s) - PAGE polyacrylamide gel electrophoresis     

Analysis of the tertiary structure of the ribonuclease P ribozyme-substrate complex by site-specific photoaffinity crosslinking.

Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein containing a catalytic RNA. The secondary structure of this ribozyme is well-established, and a low-resolution model of the three-dimensional structure of the ribozyme-substrate complex has been proposed based on site-specific crosslinking and phylogenetic comparative data [Harris ME et al., 1994 EMBO J 13:3953-3963]. However, several substructures of that model were poorly constrained by the available data. In the present analysis, additional constraints between elements within the Escherichia coli RNase P RNA-pre-tRNA complex were determined by intra- and intermolecular crosslinking experiments. Circularly permuted RNase P RNAs were used to position an azidophenacyl photoactive crosslinking agent specifically at strategic sites within the ribozyme-substrate complex. Crosslink sites were mapped by primer extension and confirmed by analysis of the mobility of the crosslinked RNA lariats on denaturing acrylamide gels relative to circular and linear RNA standards. Crosslinked species generally retained significant catalytic activity, indicating that the results reflect the native ribozyme structure. The crosslinking results support the general configuration of the structure model and predicate new positions and orientations for helices that were previously poorly constrained by the data set. The expanded library of crosslinking constraints was used, together with secondary and tertiary structure identified by phylogenetic sequence comparisons, to refine significantly the model of RNase P RNA with bound substrate pre-tRNA. The crosslinking results and data from chemical-modification and mutational studies are discussed in the context of the current structural perspective on this ribozyme.     

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     

Terbium as a solid-state probe for RNA

This paper continues previous work on the analysis of nucleic acid-terbium complexes in the solid state. The fluorescence excitation and emission spectra of the RNA-terbium(III) complex is reported. The fluorescence excitation and emission spectra of both the RNA-terbium(III) and DNA-terbium(III) complexes as trapped on millipore filters is reported. One hundred percent of the DNA combined with terbium was trapped on millipore filters. Deoxyribonucleic acid was recovered from DNA-terbium(III) complexes trapped on millipore filters using SDS-extraction. Energy transfer was shown to occur from the bases in nucleic acids to the terbium ion, whereas the actual binding of terbium to nucleic acids was due to phosphate groups. The relative fluorescence of homopolyribonucleotide-terbium complexes showed that the guanine moiety was responsible for most of the observed fluorescence. Binding studies showed an equal affinity of radioactive terbium for all the homopolyribonucleotides. The fluorescence of solid-state DNA and RNA terbium complexes was used to measure picomole quantities of DNA or RNA.     

Fluorescence as a probe for physiological integrity of freshwater cyanobacteria

The effects of energetic decoupling of phycobiliproteins (PBP) from photosystems in Nostoc sp. on the emission characteristics and fluorescence profiles of cyanobacterial photosynthetic apparatus and its components were studied using steady-state and time-resolved fluorescence emission. The steady-state measurements show a rise in fluorescence from PBP released at low ionic strength. The emission decay profile of Nostoc photosynthetic apparatus has two components with lifetimes 1.8 ns and about 0.1 ns but their relative contributions to the total emission decay vary, depending on the energetic coupling of phycobilisomes to photosystems. At low ionic strength, the contribution of the long-lived emission characteristic for free phycocyanin increased, confirming the detachment of PBP from the photosystems. We show that these effects can be used as a basis for improvement of cyanobacteria detection method. It is demonstrated that the fitting algorithm applied in the measurements with a FluoroProbe fluorometer (bbe Moldaenke, Schwentinental, Germany) can differentiate between coupled and uncoupled PBP. This approach may prove useful in monitoring the state of photosynthetic apparatus in cyanobacterial populations and their spatial distribution in water reservoirs.     

The structure of ribonuclease P protein from Staphylococcus aureus reveals a unique binding site for single-stranded RNA

Ribonuclease P (RNaseP) catalyses the removal of the 5'-leader sequence from pre-tRNA to produce the mature 5' terminus. The prokaryotic RNaseP holoenzyme consists of a catalytic RNA component and a protein subunit (RNaseP protein), which plays an auxiliary but essential role in vivo by binding to the 5'-leader sequence and broadening the substrate specificity of the ribozyme.We determined the three-dimensional high-resolution structure of the RNaseP protein from Staphylococcus aureus (117 amino acid residues) by nuclear magnetic resonance (NMR) spectroscopy in solution. The protein has an alphabeta-fold, similar to the ribonucleoprotein domain. We used small nucleic acid molecules as a model for the 5'-leader sequence to probe the propensity for generic single-stranded RNA binding on the protein surface. The NMR results reveal a contiguous interaction site, which is identical with the previously identified leader sequence binding site in RNaseP holoenzyme. The conserved arginine-rich motif does not bind single-stranded RNA. It is likely that this peptide segment binds selectively to double-stranded sections of P RNA, which are conformationally more rigid. Given the essentiality of RNaseP for the viability of the organism, knowledge of the S. aureus protein structure and insight into its interaction with RNA will help us to develop RNaseP and RNaseP protein as targets for novel antibiotics against this pathogen.     

Secondary structure formation precedes tertiary structure in the refolding of ribonuclease A.

The kinetics of refolding of ribonuclease A were monitored by circular dichroism (CD), tyrosine fluorescence and absorbance in the -40 to -10 degrees C range using a methanol cryosolvent. The native-like far-ultraviolet CD signal returned in the dead-time of the mixing, whereas the native absorbance and fluorescence signals returned in a multiphasic process at rates several orders of magnitude more slowly. Thus the secondary structure was formed much more rapidly than the tertiary structure. In addition, the absorbance signal showed evidence of an early intermediate in which one, or more, tyrosine residues was in a transiently more polar environment. A total of four kinetic phases were observed by absorbance in refolding, the slowest two of which had energies of activation consistent with proline isomerization. A refolding scheme involving initial hydrophobic collapse, concurrent with secondary structure formation, followed by much slower rearrangement to the native tertiary structure is proposed.     

The secondary structure of ribonuclease P RNA, the catalytic element of a ribonucleoprotein enzyme

Secondary structure models for the ribonuclease (RNAase) P RNAs of Bacillus subtilis and E. coli were derived by a phylogenetic comparative analysis of published sequences as well as four novel ones. The RNAase P RNA genes from Bacillus megaterium, Bacillus brevis, Bacillus stearothermophilus, and Pseudomonas fluorescens were cloned, sequenced, and compared with the other available sequences. Regions of pairing were identified by the occurrence of homologous complementary sequences that vary among the compared molecules. A common core of primary and secondary structure can be identified in all these RNAase P RNAs. The previously noted striking differences between the Bacillus and the enteric RNAase P RNAs arise not only from point mutations, but from the addition or deletion of structural domains. The primary and secondary structural features that are common to all of the RNAase P RNAs are likely to be the elements involved in the binding and cleavage of tRNA precursors, and in the interaction with the RNAase P protein.     

Enhanced RNA cleavage within bulge-loops by an artificial ribonuclease

Cleavage of phosphodiester bonds by small ribonuclease mimics within different bulge-loops of RNA was investigated. Bulge-loops of different size (1–7 nt) and sequence composition were formed in a 3′ terminal fragment of influenza virus M2 RNA (96 nt) by hybridization of complementary oligodeoxynucleotides. Small bulges (up to 4 nt) were readily formed upon oligonucleotide hybridization, whereas hybridization of the RNA to the oligonucleotides designed to produce larger bulges resulted in formation of several alternative structures. A synthetic ribonuclease mimic displaying Pyr–Pu cleavage specificity cleaved CpA motifs located within bulges faster than similar motifs within the rest of the RNA. In the presence of 10 mM MgCl₂, 75% of the cleavage products resulted from the attack of this motif. Thus, selective RNA cleavage at a single target phosphodiester bond was achieved by using bulge forming oligonucleotides and a small ribonuclease A mimic.