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At least 757 records · Page 42Linked to original sources
PubMed1992

Cellular expression of a functional nodavirus RNA replicon from vaccinia virus vectors.

RNA replication provides a powerful means for the amplification of RNA, but to date it has been found to occur naturally only among RNA viruses. In an attempt to harness this process for the amplification of heterologous mRNAs, both an RNA replicase and its corresponding RNA templates have been expressed in functional form, using vaccinia virus-bacteriophage T7 RNA polymerase vectors. Plasmids were constructed which contained in 5'-to-3' order (i) a bacteriophage T7 promoter; (ii) a full-length cDNA encoding either the RNA replicase (RNA 1) or the coat protein (RNA 2) of flock house virus (FHV), (iii) a cDNA sequence that encoded the self-cleaving ribozyme of satellite tobacco ringspot virus, and (iv) a T7 transcriptional terminator. Both in vitro and in vivo, circular plasmids of this structure were transcribed by T7 RNA polymerase to produce RNAs with sizes that closely resembled those of the two authentic FHV genomic RNAs, RNA 1 and RNA 2. In baby hamster kidney cells that expressed authentic FHV RNA replicase, the RNA 2 (coat protein) transcripts were accurately replicated. Moreover, the RNA 1 (replicase) transcripts directed the synthesis of an enzyme that could replicate not only authentic virion-derived FHV RNA but also the plasmid-derived transcripts themselves. Under the latter conditions, replicative amplification of the RNA transcripts ensued and resulted in a high rate of synthesis of the encoded proteins. This successful expression from a DNA vector of the complex biological process of RNA replication will greatly facilitate studies of its mechanism and is a major step towards the goal of harnessing RNA replication for mRNA amplification.

Animals↗
PubMed1997

Analysis of the architecture of the transcription factor sigma N (sigma 54) and its domains by circular dichroism.

Enhancer-dependent transcription in bacteria requires the alternative transcription factor sigma N (sigma 54), which forms an RNA polymerase holoenzyme that binds promoters as a transcriptionally inactive complex. We have examined the structure of sigma N by circular dichroism (CD) analysis. The sigma N protein and its domains are well structured in the absence of the core RNA polymerase subunits or promoter DNA. Denaturation of sigma N by temperature as followed by changes in CD shows a concomitant loss of secondary and tertiary structures with a melting temperature of 36 degrees C. The secondary structure displays a two-state melting curve with a second Tm of 85 degrees C. The amino-terminal Region I activation domain together with the acidic Region II does not contribute to the two-state melting. In marked contrast, the integrity of the C-terminal DNA-binding domain is required for the two-state melting. Measurements of pKb also demonstrated that a C-terminal part of sigma N, but not regions I or I + II, is required for the structural integrity of sigma N at high pH. Measurements of pKa suggested that alpha-helical structures are important in sigma N for the establishment of tertiary structural elements. The tertiary structure near ultraviolet CD signals of sigma N do not require regions I or I + II but were strongly diminished by C-terminal truncation of sigma N. Promoter DNA binding resulted in a conformational change in sigma N, permitting the determination of a binding constant. A typical B-DNA conformation was adopted by the promoter DNA. Implications for the modular domain organization of sigma N, the function of C-terminal sequences, and domain communication and its role in activation of transcription are discussed.

Base Sequence↗
PubMed1977

Analysis of polyoma virus nuclear RNA by mini-blot hybridization.

The size and sequence composition of virus-specific RNA extracted from the nuclei of mouse cells late during polyoma virus productive infection were studied by blot-hybridization analysis of 32P-labeled RNA fractionated on CH3HgOH/agarose gels. Viral RNA molecules between approximately 0.4 and 4 times the length of a complete transcript of the 5.4-kilobase circular viral DNA were found. Less than 20% of such molecules were polyadenylylated. Although viral RNA of all sizes contained species that together hybridized to the entire polyoma genome, sequences complementary to the late region were more abundant than sequences complementary to the early region in transcripts less than 10-12 kilobases long.

Base Sequence↗
PubMed1996

Circular assemblies.

During 1994 and 1995, the structures of the serum amyloid P component, the bacterial chaperonin GroEL, the 20S proteasome, the bacterial light-harvesting complexes and the tryptophan operon RNA-binding attenuation protein have been determined. These structures all form circular assemblies in which the individual subunits are related by rotational symmetry. In most cases the circular organization generates a new biophysical property and a specific biological function which have presumably been selected by evolution.

Bacterial Proteins↗
PubMed1983

Direct evidence for the preferential binding of Escherichia coli RNA polymerase holoenzyme to the ends of deoxyribonucleic acid restriction fragments.

Escherichia coli RNA polymerase holoenzyme has been observed to form a variety of nonpromoter complexes with DNA restriction fragments in experiments performed with the nitrocellulose filter assay [Melançon, P., Burgess, R. R., & Record, M. T., Jr. (1982) Biochemistry 21, 4318-4331]. Here we report the use of this assay to investigate aspects of the weak (heparin-sensitive) interactions of RNA polymerase core and holoenzyme with a 1600 base pair (bp) fragment of T7 DNA which contains no promoters or TB (tight binding; heparin-resistant) sites. Under the ionic conditions investigated [50 mM NaCl/10 mM MgCl2/10 mM sodium N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid (pH 7.7)], both core and holoenzyme bind to the linear DNA fragment and cause comparable levels of filter retention. When the DNA fragment is self-ligated into a circular molecule (nonsupercoiled), the extent of binding of holoenzyme (but not that of core) is dramatically reduced. This directly proves our previous hypotheses that holoenzyme recognizes and preferentially binds to the ends of DNA fragments and that this mode of binding is responsible for most of the heparin-sensitive filter retention of nonpromoter fragments. The residual mode of binding of holoenzyme detected with the circular DNAs was considered in determining the amount of protein bound at ends only. To calculate end-binding constants (KE), the amount of protein bound nonspecifically (which does not appear to cause efficient filter retention) was also taken into consideration. At 0 degrees C, we obtain a value for KE of (2.1 +/- 0.5) X 10(8) M-1, in good agreement with that determined earlier.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA↗
PubMed2002

Copper insertion facilitates water-soluble porphyrin binding to rA.rU and rA.dT base pairs in duplex RNA and RNA.DNA hybrids.

The binding of the copper(II) complex of water-soluble meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP) to double-helical polynucleotides has been studied by optical absorption, circular dichroism (CD), and resonance Raman spectroscopic methods. The target polymers were RNA and RNA.DNA hybrids consisting of rA.rU, rI.rC, rA.dT, and rI.dC base pairs. Relative to the metal-free H(2)TMPyP [Uno, T., Hamasaki, K., Tanigawa, M., and Shimabayashi, S. (1997) Inorg. Chem. 36, 1676-1683], CuTMPyP binds to poly(rA).poly(dT) and poly(rA).poly(rU) with a greatly increased binding constant. The external self-stacking of the porphyrin on the surface of the polymers was evident from the strong conservative-type induced CD signals. The signal intensity correlated almost linearly with the number of stacking sites on the polymer except for poly(rA).poly(dT), which showed extraordinarily strong CD signals. Thus, the bound porphyrin may impose an ordered architecture on the polymer surface, the stacking being facilitated by the more planar nature of the CuTMPyP than the nonmetal counterpart. Resonance Raman spectra of the stacked CuTMPyP were indistinguishable from those of the intercalated one with positive delta(Cbeta-H) and negative delta(Cm-Py) bending shifts, and hence the stacked porphyrins are suggested to adopt a similar structure to that of intercalated ones. Porphyrin flattening by copper insertion opens a new avenue for medical applications of porphyrins, blocking biological events related to RNA and hybrids in malignant cells.

Base Pairing↗
PubMed1985

Enhanced transcription of fibroin gene in vitro on covalently closed circular templates.

A modified method for S1 nuclease assay has been developed to quantitate an amount of radioactive transcripts in the presence of unlabeled homologous RNA. Using this method, we have assayed in vitro transcription of fibroin gene on circular and linear templates. In a posterior silk gland cell extract, covalently closed circular (ccc) DNA forms the superhelical state and supports three to 10 times more transcription than nicked circular or linear DNA does. The efficient transcription on ccc template is also observed in a middle silk gland cell extract but never seen in a HeLa cell extract. When the posterior silk gland cell extract is first incubated with pBR322 DNA and then challenged for the fibroin gene transcription on ccc DNA, the pBR322 DNA of the ccc form, but not of the linear one, inhibits the reaction. A deletion mutant carrying only 44 base pairs of the 5'-flanking sequence reserves the preference of the ccc template, but seven single-point mutants at the TATA box and -20 region abolish it. These results suggest that some components in the silk gland extract supercoil ccc DNA and stimulate the fibroin gene transcription in the absence of the upstream enhancing sequence.

Animals↗
PubMed2006

Characterization of the secondary structure and stability of an RNA aptamer that binds vascular endothelial growth factor.

Thermal denaturation studies and spectroscopic studies were employed to investigate the secondary structure and stability of an RNA-PEG conjugate commercially called Macugen. The RNA aptamer is conjugated to a pegylated moiety, and the majority of its 2'-hydroxyl groups are methylated or otherwise modified. UV optical melting studies and differential scanning calorimetry (DSC) were carried out under different conditions to evaluate the effects of Na+ and oligomer concentrations on the stability of the secondary structure of the RNA oligomer. The results of these studies indicated that the T(m) of the RNA is independent of oligomer concentration but dependent on the salt concentration, in a predictable fashion. Further, the DSC melting profiles obtained under all conditions were highly reversible. Circular dichroism (CD) studies were determined under different salt concentrations, various RNA concentrations, and temperatures as well. Together, the thermal denaturation and CD studies provide evidence that the secondary structure of the RNA oligonucleotide is a stable hairpin at 25 degrees C and that the thermally induced hairpin to single strand transition is highly reversible.

Aptamers, Nucleotide↗
PubMed2001

Excision of group II introns as circles.

Group II introns are usually removed from precursor RNAs as lariats comprised of a circular component and a short 3' tail. We find that group II introns can also be excised as complete circles. Circle formation requires release of the 3' exon of a splicing substrate, apparently by a trans splicing mechanism. After 3' exon release, the terminal uridine of the intron attacks the 5' splice site, releasing the 5' exon and joining the first and last intron residues by a 2'-5' phosphodiester bond. RNA isolated from yeast mitochondria also contains circles, indicating that at least one group II intron (aI2) forms circles in vivo. Furthermore, analysis of RNA and DNA from certain mutant yeast strains shows that circular DNA introns exist and are produced by reverse transcription of RNA, rather than by ectopic homing.

DNA Ligases↗
PubMed2006

Synthesis and properties of 4'-ThioDNA: unexpected RNA-like behavior of 4'-ThioDNA.

The synthesis and properties of fully modified 4'-thioDNAs, oligonucleotides consisting of 2'-deoxy-4'-thionucleosides, were examined. In addition to the known literature properties (preferable hybridization with RNA and resistance to endonuclease hydrolysis), we also observed higher resistance of 4'-thioDNA to 3'-exonuclease cleavage. Furthermore, we found that fully modified 4'-thioDNAs behaved like RNA molecules in their hybridization properties and structural aspect, at least in the case of the 4'-thioDNA duplex. This observation was confirmed by experiments using groove binders, in which a 4'-thioDNA duplex interacts with an RNA major groove binder, lividomycin A, but not with DNA groove binders, to give an increase in its thermal stability. Since a 4'-thioDNA duplex competitively inhibited the hydrolysis of an RNA duplex by RNase V1, it was not only the physical properties but also this biological data suggested that a 4'-thioDNA duplex has an RNA-like structure.

Circular Dichroism↗
PubMed1999

Folding of a large ribozyme during transcription and the effect of the elongation factor NusA.

We compared in vitro transcription-initiated folding of the ribozyme from Bacillus subtilis RNase P to refolding from the full-length, denatured state by monitoring the appearance of its catalytic activity. At 37 degrees C, Mg(2+)-initiated refolding of the wild type and a circularly permutate ribozyme takes minutes and is limited by a kinetic trap. Transcription by T7 RNA polymerase alters the folding pathway of both RNAs and introduces new kinetic traps. Transcription by the core Escherichia coli RNA polymerase yields the same result, in spite of its 4-fold-slower elongation rate. However, the presence of its elongation factor NusA accelerates more than 10-fold the transcription-initiated folding of the circularly, permutated ribozyme by E. coli RNA polymerase. The effect of NusA likely is caused by its enhancement of transcriptional pausing because NusA did not accelerate transcription-initiated folding using a mutant RNA polymerase that failed to pause or respond to NusA during ribozyme synthesis. We conclude that both transcription and specific pausing therein can alter RNA-folding pathways.

Bacillus subtilis↗
PubMed2004

Effect of LNA modifications on small molecule binding to nucleic acids.

Locked nucleic acid (LNA) is a conformationally constrained DNA analogue that exhibits exceptionally high affinity for complementary DNA and RNA strands. The deoxyribose sugar is modified by a 2'-O, 4'-C oxymethylene bridge, which projects into the minor groove. In addition to changing the distribution of functional groups in the groove and the overall helical geometry relative to unmodified DNA, the bridge likely alters the hydration of the groove. Each of these factors will impact the ability of small molecules, proteins and other nucleic acids to recognize LNA-containing hybrids. This report describes the ability of several DNA-intercalating ligands and one minor groove binder to recognize LNA-DNA and LNA-RNA hybrid duplexes. Using UV-vis, fluorescence and circular dichroism spectroscopies, we find that the minor groove binder as well as the intercalators exhibit significantly lower affinity for LNA-containing duplexes. The lone exception is the alkaloid ellipticine, which intercalates into LNA-DNA and LNA-RNA duplexes with affinities comparable to unmodified DNA-DNA and RNA-DNA duplexes.

Bisbenzimidazole↗
PubMed1973

A study of the thermal stability of ribosomes and biologically active subribosomal particles.

1. The ability of Escherichia coli ribosomes to function in poly(U)-directed protein synthesis was measured at elevated temperatures by using thermostable supernatant factors from Bacillus stearothermophilus. The amount of polyphenylalanine synthesized at 55 degrees C was about the same as at 37 degrees C, but the rate of synthesis was increased approximately fivefold. At 60 degrees C the activity of the ribosomes was halved. 2. E. coli ribosomes can sustain the loss of approx. 10% of the double-helical secondary structure of RNA without losing activity. 3. Within the active ribosome the double-helical secondary structure of the rRNA moiety is stabilized compared with isolated rRNA, as judged by enzymic hydrolysis and by measurements of E(260). 4. The main products, over the range 0-55 degrees C, of ribonuclease T(1) digestion of the smaller subribosomal particle of E. coli were two fragments (s(0) (20,w) 15S and 25.3S) of approximately one-quarter and three-quarters of the size of the intact molecule, revealing the presence of a ;weak spot' where intramolecular bonds appear insufficient to hold the fragments together. 5. Subribosomal particles of B. stearothermophilus were more stable to heating, by approx. 10 degrees C, than those of E. coli, and the stabilization of double-helical secondary structure of the RNA moiety was more striking. 6. Rabbit reticulocyte ribosomes were active in poly(U)-directed protein synthesis at 45 degrees C, and half the activity was lost after heating to 53 degrees C. Active subribosomal particles of rabbit reticulocytes and of oocytes of Xenopus laevis, like the bacterial subribosomal particles, underwent a conformational change to a slower-sedimenting form on heating. The temperature range of the transition depended on the species. 7. Slower-sedimenting particles, whether produced by EDTA treatment or by heating, had different ;melting' profiles compared with active subribosomal particles, providing another indication of conformational differences. 8. Comparison of the properties of the various subribosomal particles revealed greater variation in the secondary structure of the protein moieties (judged by measurement of circular dichroism) than in the secondary structure of the RNA moieties, which appeared to have features in common.

Animals↗
PubMed1995

Interaction of methylene blue with transfer RNA--a spectroscopic study.

The binding of methylene blue (MB) with tRNA was studied using absorption, fluorescence and circular dichroic spectroscopy. In the spectral titration of MB with tRNA, hypochromism was observed in the absorption maximum of the dye in the visible region till P/D = 4 and thereafter the intensity increased with a red shift at P/D > 9, indicating electrostatic and intercalative binding at low and high P/D ratios, respectively. Analysis of absorption data, following Schwarz's procedure, showed that the electrostatic binding is cooperative in nature (cooperatively parameter q = 50) with a binding constant K = 7.77 x 10(3) M-1. A non-linear Scatchard plot was observed for the intercalative binding (at P/D > 4), probably due to a difference in the spectral characteristics of the dye intercalated between the base pairs and that between the bases in the single stranded domains. Quenching of fluorescence was observed for both the binding processes. In the circular dichroism spectra of tRNA-MB complexes at high P/D (approximately 30), nonconservative positive ICD bands were seen at 620 and 680 nm while at low P/D (approximately 2), two conservative negative CD bands at 300 and 660 nm and two bisignate bands with cross overs at 565 and 605 nm were observed. The short wavelength component of the bisignate band at 565 nm is negative while that of the 605-nm band is positive, indicating that the former arises from left handed and the latter from the right handed helical disposition of dye molecules along the tRNA backbone. The changes in the CD spectrum of tRNA on dye binding could be due to a conformational change of the nucleic acid or a negative CD being induced at that region.

Circular Dichroism↗
PubMed1996

Comparison of the thermodynamic stabilities and solution conformations of DNA.RNA hybrids containing purine-rich and pyrimidine-rich strands with DNA and RNA duplexes.

The conformations and thermodynamic stabilities of duplexes containing purine-rich (GAA-GAGAAGC) and pyrimidine-rich (GCTTCTCTTC or GCUUCUCUUC) DNA and RNA strands have been measured by UV melting, electrophoresis, circular dichroism, and NMR spectroscopy. The free energies of stabilization (-delta G) were in the order rR.rY > rR.dY > dR.dY > dR.rY. The two DNA.RNA hybrid duplexes showed conformational properties intermediate between those of DNA.DNA and RNA.RNA duplexes and also different from one another. Differences between 1H chemical shifts of the DNA strands in the two hybrid duplexes and those of the DNA duplex were larger than analogous shift differences for the RNA protons, and the differences were larger for the purine than the pyrimidines in the DNA strands. Detailed analysis of the nucleotide conformations using both NOE and scalar coupling data showed that the sugar conformations of the ribonucleotides are all near C3'-endo. The deoxyribonucleotides were in the "S" domain, i.e., near C2'-endo in the DNA duplex, and C1'-exo to C2'-endo in the two hybrids. However, the deoxyriboses in the two hybrids appear more flexible than in the DNA duplex, with the fraction in the "N" (C3'-endo) state increasing in the order dR.dY < dR.rY < rR.dY. Globally, the pure DNA duplex was B-form and the pure RNA duplex A form. The two DNA.RNA hybrids were neither A nor B, but closer globally to the A than the B form. The less stable dR.rY duplex has a significantly different conformation from rR.dY both at the local nucleotide level and globally.

Carbohydrate Conformation↗
PubMed2002

Isolation and properties of noncovalent complex of transketolase with RNA.

A method for isolation of homogenous transketolase from baker's yeast using immunoaffinity chromatography was significantly simplified. It was demonstrated that transketolase could be isolated from fresh yeast in the form of a complex with a high molecular weight RNA. Storage of yeast led to the dissociation of the complex to a low molecular weight complex and then to the free enzyme. Conditions were chosen for complex dissociation and free enzyme isolation. In comparison to the free enzyme, the specific activities of the high and low molecular weight complexes were decreased 20-25- and 3-5.5-fold, respectively. The affinity to the cofactor thiamine diphosphate and to xylulose-5-phosphate (donor substrate) did not change for the low molecular weight complex, while the time of binding to calcium increased. The latter was necessary for the complete manifestation of the enzymatic activity. Changes in the circular dichroism spectrum between 300 and 360 nm after the addition of thiamine diphosphate, which characterize the formation of the catalytically active holoenzyme, were significantly lower for the low molecular weight complex than for the free enzyme.

Circular Dichroism↗