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At least 847 records · Page 47Linked to original sources
PubMed1987

DNA bound RNA polymerase activity of the yeast nuclei.

A preparation of nuclei from yeast cells is shown to be capable of autodigesting its chromatin in the presence of a series of magnesium containing buffers having different concentrations of potassium. The 'digests' or 'extracts' prepared in this manner contain an active transcription complex which can transcribe endogenous as well as exogenous DNA template. The effects of heparin and actinomycin D on the in vitro transcription by these complexes confirm the presence of RNA polymerase in DNA bound form. Further purification of this transcription complex is attempted by sucrose density gradient centrifugation. Circular dichroic measurements show that the nuclear DNA exists in compact state in the extracts.

Cell Nucleus↗
PubMed1998

Expression, purification and characterization of the recombinant ribonuclease P protein component from Bacillus subtilis.

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.

Amino Acid Sequence↗
PubMed1997

Design and analysis of molecular motifs for specific recognition of RNA.

Selective targeting of RNA has become a recent priority in drug design strategies due to the emergence of retroviruses, the need for new antibiotics to counter drug resistance, and our increased awareness of the essential role RNA and RNA structures play in the progression of disease. Most organic compounds known to specifically target RNA are complex, naturally occurring antibiotics that are difficult to synthesize or derivatize and modification of these compounds to optimize interactions with structurally unique RNAs is difficult. The de novo design of synthetically accessible analogues is one possible alternative; however, little is known about the RNA recognition principles on which to design new compounds and limited information on RNA structure in general is available. To contribute to the growing body of knowledge on RNA recognition principles, we have prepared two series of polycationic RNA-binding agents, one with a linear scaffold, the other with a macrocyclic scaffold. We evaluated these compounds for their ability to bind to DNA and RNA, as well as to a specific RNA, the regulatory sequence, RRE, derived from HIV-1, by using thermal melting, circular dichroism, and electrophoresis gel shift methods. Out results suggest that cationic charge centers of high pKa that are displayed along a scaffold of limited flexibility bind preferentially to RNA, most likely within the major groove. Related derivatives that bind more strongly to DNA more closely mimic classical DNA minor-groove binding agents. Several of the macrocyclic polycations expand on a new binding motif where purine bases in duplex RNA are complexed within the macrocyclic cavity, enhancing base-pair opening processes and ultimately destabilizing the RNA duplex. The results in this report should prove a helpful addition to the growing information on molecular motifs that specifically bind to RNA.

Amino Acid Sequence↗
PubMed1988

Viral RNA polymerases.

Recent progress in molecular biological techniques revealed that genomes of animal viruses are complex in structure, for example, with respect to the chemical nature (DNA or RNA), strandedness (double or single), genetic sense (positive or negative), circularity (circle or linear), and so on. In agreement with this complexity in the genome structure, the modes of transcription and replication are various among virus families. The purpose of this article is to review and bring up to date the literature on viral RNA polymerases involved in transcription of animal DNA viruses and in both transcription and replication of RNA viruses. This review shows that the viral RNA polymerases are complex in both structure and function, being composed of multiple subunits and carrying multiple functions. The functions exposed seem to be controlled through structural interconversion.

DNA-Directed RNA Polymerases↗
PubMed2000

Synthesis of aminoglycoside-modified oligonucleotides.

[structure: see text] To study the structural requirements of aminoglycoside binding to nucleic acids, compound 1-an analogue of the naturally occurring nucleoside J-was synthesized. When incorporated into oligodeoxynucleotides, 1 leads to thermal stabilization of the resulting duplexes. The increase in pairing affinity is stronger with complementary RNA than with DNA.

Aminoglycosides↗
PubMed1998

Visualization of the binding site for the transcript cleavage factor GreB on Escherichia coli RNA polymerase.

The structure of Escherichia coli core RNA polymerase (RNAP) complexed with the transcript cleavage factor GreB was determined from electron micrographs of negatively stained, flattened helical crystals. A binding assay was developed to establish that GreB was incorporated into the RNA polymerase crystals with high occupancy through interactions between the globular C-terminal domain and the RNA polymerase. Comparison of the core RNAP:GreB structure with the previously determined structure of core RNAP located the GreB binding site on one face of the RNA polymerase, next to but not in the 25 A-diameter channel of RNA polymerase.

Binding Sites↗
PubMed2006

Analogues of a locked nucleic acid with three-carbon 2',4'-linkages: synthesis by ring-closing metathesis and influence on nucleic acid duplex stability and structure.

Two bicyclic 2'-deoxynucleoside analogues containing a saturated and an unsaturated three-carbon 2',4'-linkage, respectively, have been synthesized using a ring-closing metathesis-based linear strategy starting from uridine. Both analogues have been incorporated into oligodeoxynucleotide sequences and increased the stability of DNA:RNA hybrid duplexes (DeltaT(m) approximately 2.5-5.0 degrees C per modification) and decreased the stability of dsDNA duplexes (DeltaT(m) approximately 2.5-1.0 degrees C per modification). CD spectroscopy revealed that the bicyclic nucleosides induced formation of A-type-like duplexes albeit to a lesser degree than found for locked nucleic acid (LNA) monomers. From the CD data and UV melting analysis, we propose that the 2'-oxygen atom of the bicyclic moiety is essential for the formation of stabilized A-type-like dsDNA but not for the formation of a stabilized A-type DNA:RNA hybrid.

Base Sequence↗
PubMed2002

Flavin recognition by an RNA aptamer targeted toward FAD.

Flavin adenine dinucleotide (FAD) is one of the primary cofactors in biological redox reactions. Designing cofactor-dependent redox ribozymes could benefit from studies of new RNA-cofactor complexes, as would our understanding of ribozyme evolution during an RNA World. We have therefore used the SELEX method to identify RNA aptamers that recognize FAD. Functional analysis of mutant aptamers, S1 nuclease probing, and comparative sequence analysis identified a simple, 45 nt helical structure with several internal bulges as the core-binding element. These aptamers recognize with high specificity the isoalloxazine nucleus of FAD but do not distinguish FAD from FADH(2), nor are they removed from an FAD resin with UMP (which shares a pattern of hydrogen bond donors and acceptors along one face). Thus, these aptamers are structurally and functionally distinct from previously identified FMN and riboflavin aptamers. Circular dichroism data suggest a conformational change in the RNA upon FAD binding. These aptamers require magnesium and are active across a wide pH range (4.5-8.9). Since general acid-base catalysis plays a role in some flavin-dependent redox reaction mechanisms, these aptamers may be particularly well-suited to the design of new redox ribozymes.

Base Sequence↗
PubMed1998

Initiation of mitochondrial DNA replication by transcription and R-loop processing.

The mitochondrial genome of eukaryotic cells is maintained by a mechanism distinct from that employed in the nucleus. Mitochondrial DNA replication at the leading-strand origin is coupled to transcription through the formation of an RNA-DNA hybrid known as an R-loop. In vivo and in vitro evidence has implicated an RNA processing enzyme, RNase MRP, in primer maturation. In our investigation of mammalian RNase MRP, we have analyzed its specific endoribonuclease activity on model R-loops. We demonstrate here that human RNase MRP cleaves this distinctly configured substrate at virtually all of the major DNA replication sites previously mapped in vivo. We further show that the processed RNA products remain stably base-paired to the template DNA strand and are functional for initiating DNA synthesis on a closed circular plasmid. Thus, in vitro initiation of leading-strand mtDNA synthesis requires only the actions of RNA polymerase and RNase MRP for the generation of replication primers.

Base Sequence↗
PubMed1978

Inverted complementary terminal sequences in single-stranded RNAs and snap-back RNAs from vesicular stomatitis defective interfering particles.

Complementary single-stranded RNAs from three independent VSV defective interfering particle (DI) sources examined can anneal and give rise to monomeric and multimeric circular and linear double-stranded structures observable by electron microscopy under aqueous conditions. When the RNA from the shortest of these DI is spread from 80% formamide solutions, as many as 32% of the molecules are circular, suggesting that the single-stranded RNAs contain inverted complementary terminal sequences. This is strongly supported by the isolation of the putative terminal sequences which rapidly become RNase resistant base-paired structures after melting and quick-cooling the RNA. RNase digestion yields a major and a minor component, 60 to 70 and 135 to 170 nucleotides long respectively. Snap-back DI RNAs also contain inverted complementary sequences at both ends of the plus and minus strands of the duplexes since nicking these at the ends gives rise to double-stranded molecules which can form monomeric and multimeric circular and linear molecules. Thus, snap-back molecules most likely contain a covalent linkage between or near complementary terminal sequences on the two complementary strands as schematically shown in Fig. 5D.

Base Sequence↗
PubMed1994

Thermodynamic stability and solution conformation of tandem G.A mismatches in RNA and RNA.DNA hybrid duplexes.

G.A mismatches form a variety of hydrogen-bonded structures in DNA, most of which destabilise the duplex. Tandem G.A mismatches in the context YGAR (Y = pyrimidine, R = purine), however, form base pairs using the amino group of the guanine residue [Li, Y., Zon. G. & Wilson, W.D. (1991) Proc. Natl Acad. Sci. USA 88, 26-30], which permits extensive base-base stacking, leading to a slight stabilisation of the helix [Ebel, S., Lane, A. N. & Brown, T. (1992) Biochemistry 31, 12083-12086]. We have measured the thermodynamic stability of several RNA and RNA.DNA hybrid duplexes containing tandem G.A mismatches. The RNA duplexes are intrinsically much more stable than the corresponding DNA duplexes and the mutations are destabilising in all cases. NOE and coupling-constant data show that all of the sugars are in the C3'-endo range of conformations, and glycosidic torsion angles are in the range -160 degrees to -180 degrees in [sequence: see text]. Both sequential NOE intensities and circular-dichroism measurements indicate that the global conformation of the mismatched RNA is A-like. The N1H group of the mismatched guanine residue is not involved in hydrogen bonding with the adenine residue, indicating the presence of the amino-pairing scheme. Determination of the structure using 'loose' NMR-derived constraints shows that the potential energies of the imino-paired and amino-paired forms are similar, but substantially higher than energy-minimised RNA. Using tighter constraints derived from more extensive analysis of one-dimensional and two-dimensional NOE data showed that the amino-paired structure agrees with the constraint data better than the imino-paired structure, and also accounts for unusual chemical shifts and the lack of hydrogen bonding of the guanine N1H group. Resulting molecular models show that the amino-paired mismatches are not as extensively stacked on the neighbouring part of the duplex as in the B-DNA analogues, largely accounting for the lower thermodynamic stability in the RNA duplexes.

Base Sequence↗
PubMed1991

Selectivity of Escherichia coli RNA polymerase for template conformation.

Escherichia coli RNA polymerase (RNAP) exhibits a strong selectivity for the secondary structure of its template DNA, as shown by the influence both of the DNA conformation on the transcription cycle and of the enzyme on the DNA conformation itself. Binding, chain initiation and elongation characteristics of RNAP, and DNA conformational characteristics were examined by use of the alternating copolymer poly(dGdm5C).poly(dGdm5C) as template. Transcription is impeded when the DNA is in the Z conformation as compared with the B; the initial conformation is determined by the concentration of the conformational effectors of Mg2+ and [Co(NH3)6]3+. RNAP binds to both Z and B conformers; the total binding is moderately greater when the template is in the B conformation than when it is strongly stabilized in the Z, by [Co(NH3)6]3+ concentrations much higher than those required for B-Z transition. However, the Z conformer is much more easily displaced competitively from the bulk of its complexes with RNAP than is the B, indicating a specific binding preference for the B conformer. When the template is in the B conformation, or is moderately stabilized in the Z by Mg2+ concentrations such that the polynucleotide is just fully converted from B to Z, elongation is predicted well by chain initiation, indicating that on the Z conformer RNAP is effectively inhibited at the chain initiation or at an earlier stage. The average chain growth rates for polymeric product synthesized on B and on moderately stabilized Z are similar, even though overall RNA synthesis is considerably lowered on the Z form, again indicating that the limiting events precede elongation. When the Z conformer is strongly stabilized, chain initiation and elongation are further inhibited. Elongation is still roughly correlated with chain initiation, but some additional inhibition of elongation takes place independently. Circular dichroism analysis shows that RNAP-DNA binding affects the B-Z conformational equilibrium, leading to reformation of the B conformer from Z and interference with conversion of B to Z, under conditions that would otherwise favor the Z conformer. Thus, there is an RNAP concentration dependent shift of the B-Z transition to higher concentrations of Z-inducing cation, and there is an RNAP concentration dependent decrease in the rate of B to Z conversion. These effects were observed for poly(dGdm5C).poly(dGdm5C), with Z stabilized by [Co(NH3)6]3+ or Mg2+. (They were observed as well for the unmethylated copolymer poly(dGdC).poly(dGdC), with Z stabilized by [Co(NH3)6]3+.) Perturbation of the Z conformer was detectable by circular dichroism at an RNAP:polynucleotide ratio down to a practical limit of approximately 1 RNAP:500 bp.(ABSTRACT TRUNCATED AT 400 WORDS)

Circular Dichroism↗
PubMed1996

Transcript mapping and processing of mitochondrial RNA in the chlorophyte alga Prototheca wickerhamii.

The detailed transcript map of the circular 55328 bp mitochondrial (mt) genome from the colourless chlorophycean alga Prototheca wickerhamii has been determined. On each half of this genome the genes are encoded only on one DNA strand, forming transcriptional units comprising variable numbers of genes. With the exception of four genes coding for ribosomal proteins, transcripts of the three rRNA genes and all protein-coding genes have been detected by both northern analysis and primer extension experiments. Polycistronic transcripts of protein coding and tRNA genes were verified by northern analyses, primer extension and RNAse mapping experiments. The 5' and 3' ends of different RNA species are often located in close proximity to putative stem-loop structures and some 5' termini of mRNAs coincide with the 3' end of tRNAs located immediately upstream. Transcript mapping in a putative promoter region revealed two different possible transcription initiation sites; no significant sequence homology to putative mt promoters from higher plants could be found. In addition, two out of three group I introns residing in the cox1 gene were found to be self-splicing in vitro under reaction conditions developed for related mt introns from a filamentous fungus. Mitochondrial gene expression of P. wickerhamii and of filamentous fungi has several features in common, such as intron splicing and the processing of longer polycistronic transcripts. The similarities in RNA maturation between higher-plant and P. wickerhamii mitochondria are less pronounced, since plants rarely use tRNAs as processing signals for their relatively short mitochondrial co-transcripts.

Base Sequence↗