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PubMed2001

Structural properties of carnation mottle virus p7 movement protein and its RNA-binding domain.

Plant viral movement proteins (MPs) participate actively in the intra- and intercellular movement of RNA plant viruses to such an extent that MP dysfunction impairs viral infection. However, the molecular mechanism(s) of their interaction with cognate nucleic acids are not well understood, partly due to the lack of structural information. In this work, a protein dissection approach was used to gain information on the structural and RNA-binding properties of this class of proteins, as exemplified by the 61-amino acid residue p7 MP from carnation mottle virus (CarMV). Circular dichroism spectroscopy showed that CarMV p7 is an alpha/beta RNA-binding soluble protein. Using synthetic peptides derived from the p7 sequence, we have identified three distinct putative domains within the protein. EMSA showed that the central region, from residue 17 to 35 (represented by peptide p7(17-35)), is responsible for the RNA binding properties of CarMV p7. This binding peptide populates a nascent alpha-helix in water solution that is further stabilized in the presence of either secondary structure inducers, such as trifluoroethanol and monomeric SDS, or RNA (which also changes its conformation upon binding to the peptide). Thus, the RNA recognition appears to occur via an "adaptive binding" mechanism. Interestingly, the amino acid sequence and structural properties of the RNA-binding domain of p7 seem to be conserved among carmoviruses and some other RNA-binding proteins and peptides. The low conserved N terminus of p7 (peptide p7(1-16)) is unstructured in solution. In contrast, the highly conserved C terminus motif (peptide p7(40-61)) adopts a beta-sheet conformation in aqueous solution. Alanine scanning mutagenesis of the RNA-binding motif showed how selected positive charged amino acids are more relevant than others in the RNA binding process and how hydrophobic amino acid side chains would participate in the stabilization of the protein-RNA complex.

Amino Acid Sequence↗
PubMed2000

Kir3.1/3.2 encodes an I(KACh)-like current in gastrointestinal myocytes.

Expression of the Kir3 channel subfamily in gastrointestinal (GI) myocytes was investigated. Members of this K(+) channel subfamily encode G protein-gated inwardly rectifying K(+) channels (I(KACh)) in other tissues, including the heart and brain. In the GI tract, I(KACh) could act as a negative feedback mechanism to temper the muscarinic response mediated primarily through activation of nonselective cation currents and inhibition of delayed-rectifier conductance. Kir3 channel subfamily isoforms expressed in GI myocytes were determined by performing RT-PCR on RNA isolated from canine colon, ileum, duodenum, and jejunum circular myocytes. Qualitative PCR demonstrated the presence of Kir3.1 and Kir3.2 transcripts in all smooth muscle cell preparations examined. Transcripts for Kir3.3 and Kir3.4 were not detected in the same preparations. Semiquantitative PCR showed similar transcriptional levels of Kir3.1 and Kir3.2 relative to beta-actin expression in the various GI preparations. Full-length cDNAs for Kir3.1 and Kir3.2 were cloned from murine colonic smooth muscle RNA and coexpressed in Xenopus oocytes with human muscarinic type 2 receptor. Superfusion of oocytes with ACh (10 microM) reversibly activated a Ba(2+)-sensitive and inwardly rectifying K(+) current. Immunohistochemistry using Kir3.1- and Kir3.2-specific antibodies demonstrated channel expression in circular and longitudinal smooth muscle cells. We conclude that an I(KACh) current is expressed in GI myocytes encoded by Kir3.1/3.2 heterotetramers.

Acetylcholine↗
PubMed2005

Arginine-rich motifs present multiple interfaces for specific binding by RNA.

A number of proteins containing arginine-rich motifs (ARMs) are known to bind RNA and are involved in regulating RNA processing in viruses and cells. Using automated selection methods we have generated a number of aptamers against ARM peptides from various natural proteins. Aptamers bind tightly to their cognate ARMs, with K(d) values in the nanomolar range, and frequently show no propensity to bind to other ARMs or even to single amino acid variants of the cognate ARM. However, at least some anti-ARM aptamers can cross-recognize a limited set of other ARMs, just as natural RNA-binding sites have been shown to exhibit so-called "chameleonism." We expand upon the number of examples of cross-recognition and, using mutational and circular dichroism (CD) analyses, demonstrate that there are multiple mechanisms by which RNA ligands can cross-recognize ARMs. These studies support a model in which individual arginine residues govern binding to an RNA ligand, and the inherent flexibility of the peptide backbone may make it possible for "semi-specific" recognition of a discrete set of RNAs by a discrete set of ARM peptides and proteins.

Alanine↗
PubMed1980

Melting of Saccharomyces cerevisiae 5S ribonucleic acid: ultraviolet absorption, circular dichroism, and 360-MHz proton nuclear magnetic resonance spectroscopy.

The heat-induced melting of yeast 5S RNA and tRNAPhe has been monitored by UV, CD, and 360-MHz 1H NMR spectroscopy in order to determine the extent of base stacking and base pairing in the native and denatured structures. In the presence of Mg2+, the optical data indicate less than or equal to 40 base pairs in native yeast 5S RNA, a 60:40 ratio of GC to AU base pairs, with more single-stranded stacking and a slightly less stable structure (half-melted at 67 degrees C) than for tRNAPhe (half-melted at 71 degrees C). In the absence of Mg2+, the NMR results identify a minimum of approximately 32 base pairs at 25 degrees C (increasing to a minimum of approximately 35 base pairs in the presence of Mg2+), of which more than half are still intact at 48 degrees C. The native structure (25 degrees C) shows only minor dependence upon Mg2+ concentration, and no denatured forms could be detected. Finally, the present results support a previously proposed cloverleaf secondary structure for eukaryotic 5S RNA.

Base Composition↗
PubMedDate not supplied

[Circular dichroism spectra study on the ribonucleoprotein structure of influenza virus].

The CD spectra of Influenza virus RNA and RNP were examined in the ultraviolet region (220--320 nm) at different temperatures, NaCl and urea concentrations. The magnitude of the positive band for RNP decreases gradually upon increasing the temperature, indicating that rather weak interaction between RNA and protein occurs. However the temperature drastically affects the intensity of the negative band about 222 nm. In the temperature range where the protein melts the positive band at longer wavelengths shows a temperature dependence that is similar for RNP and free RNA. Addition of NaCl results in an increase in intensity and blue shift of the positive CD band of RNP. In the presence of 1 M NaCl the CD spectrum of RNP is very close to that of protein-free RNA. Urea concentrations up to 10 M has little effect on the CD spectrum of RNP. These results suggest that only ionic but not hydrophobic and hydrogen bondings are involved in the RNA-protein interaction in Influenza virus RNP.

Circular Dichroism↗
PubMed1989

Transcriptional mapping of the amplified region encoding the dihydrofolate reductase-thymidylate synthase of Leishmania major reveals a high density of transcripts, including overlapping and antisense RNAs.

We have examined the transcriptional organization of the R region of the protozoan parasite Leishmania major. This region encodes the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) and is frequently amplified as a 30-kilobase (kb) extrachromosomal circular DNA in methotrexate-resistant lines. Northern (RNA) blot analysis shows that the R region encodes at least 10 stable cytoplasmic polysomal poly(A)+ RNAs, ranging in size from 1.7 to 13 kb and including the 3.2-kb DHFR-TS mRNA. Transcriptional mapping reveals that these RNAs are closely spaced and collectively cover more than 95% of the 30-kb amplified R region. The organization is complex, including several overlapping RNAs 3' of DHFR-TS and two examples of antisense RNAs 5' of DHFR-TS. The R region RNAs can be grouped into two empirical domains, with eight contiguous RNAs transcribed in the same direction as that of DHFR-TS and two contiguous RNAs transcribed in the orientation opposite to that of DHFR-TS. The two 5'-most RNAs of the DHFR-TS-containing domain overlap the RNAs transcribed from the opposite strand. These data are relevant to models of transcription, including recent studies suggesting polycistronic transcription in trypanosomatids. The abundance of R region RNAs increases uniformly 10- to 15-fold in the amplified R1000-3 line relative to the wild type, and no new RNAs were observed. This suggests that all elements required in cis for DHFR-TS expression are contained within the 30-kb circular DNA. Quantitative analysis reveals that the steady-state DHFR-TS mRNA and protein levels are not growth phase regulated, unlike the monofunctional mouse DHFR. DHFR-TS is developmentally regulated, however, declining about fivefold in lesion amastigotes relative to promastigotes.

Animals↗
PubMed1986

Purified Saccharomyces cerevisiae RNA polymerase II interacts homologously with two different promoters as revealed by P1 endonuclease analysis.

The intergenic region of the Saccharomyces cerevisiae GAL1-GAL10 divergent promoters has been circularized in vitro in different topological states. In defined conditions, purified homologous RNA polymerase II forms two stable complexes (half-life approximately equal to 5 h) with this DNA in the presence of the four ribonucleotides, as determined by measurement (Gamper and Hearst 1983) of the amount and stability of the resulting unwinding. Each stable complex induces in the closed DNA domain a region of hypersensitivity to P1 endonuclease. The two induced hypersensitive regions are very similar: each maps on one promoter, spans over the 100 bp DNA sequence that encompasses the RNA Initiation Sites (RIS) and the TATA box, is composed by three subregions (one on the RIS, one proximal or overlapping the TATA sequence, one intermediate). We show that this promoter-localized interaction is supercoil-dependent.

Base Sequence↗
PubMed1978

Structural changes of ribosome by the action of ethylene glycol.

The denaturation of ribosome and RNA by ethylene glycol (EG) has been studied in an attempt to further understand the conformation and stability of the ribosome. At high concentrations of EG, the ribosome, its subunits, and 16S RNA undergo drastic structural changes as shown by circular dichroism, ultraviolet absorption spectroscopy, and sedimentation velocity. Two separate conformational transitions were observed for the 30S subunit; one from 30 to 50% EG and another from 60 to 90% EG. This observation suggests the presence of two "domains" in the 30S subunit which differ in their stability. However, the 50S subunit undergoes a single sharp transition at 60 to 90% EG, consistent with the notion of a highly cooperative conformation. Association of the subunits stablizes part of the 30S subunit since the transition curve for the 70S ribosome does not exhibit significant change at the low EG concentration region as seen for the 30S subunit. Removal of proteins from the 30S subunit broadens the transition curve to lower EG concentrations and suggests the role of proteins in stabilizing the conformation of the 16S RNA.

Cell Fractionation↗
PubMed1982

Nucleotide sequence and secondary structure of citrus exocortis and chrysanthemum stunt viroid.

The complete nucleotide sequence of citrus exocortis viroid (CEV, propagated in Gymura) and chrysanthemum stunt viroid (CSV, propagated in Cineraria) has been established, using labelling in vitro and direct RNA sequencing methods and a new screening procedure for the rapid selection of suitable RNA fragments from limited digests. The covalently closed circular single-stranded viroid RNAs consist of 371 (CEV) and 354 (CSV) nucleotides, respectively. As previously shown for potato spindle tuber viroid (PSTV, 359 nucleotides), CEV and CSV also contain a long polypurine sequence. Maximal base-pairing of the established CEV and CSV sequences results in an extended rod-like secondary structure similar to that previously established for PSTV and as predicted from detailed physicochemical studies of all these viroids. Although the three viroid species sequenced to date differ in size and nucleotide sequence, there is 60--73% homology between them. As PSTV, CEV and CSV also contain conserved complementary sequences which are separated from each other in the native secondary structure. We postulate that the resulting 'secondary' hairpins, being formed and observed in vitro during the complex process of thermal denaturation of viroid RNA, must have a vital, although yet unknown, function in vivo. The possible origin and function of viroids are discussed on the basis of the characteristic structural features and of a considerable homology with U1a RNA found for a region highly conserved in the three viroids.

Base Sequence↗
PubMed2006

Transcription of potato spindle tuber viroid by RNA polymerase II starts in the left terminal loop.

Viroids are single-stranded, circular RNAs of 250 to 400 bases, that replicate autonomously in their host plants but do not code for a protein. Viroids of the family Pospiviroidae, of which potato spindle tuber viroid (PSTVd) is the type strain, are replicated by the host's DNA-dependent RNA polymerase II in the nucleus. To analyze the initiation site of transcription from the (+)-stranded circles into (-)-stranded replication intermediates, we used a nuclear extract from a non-infected cell culture of the host plant S. tuberosum. The (-)-strands, which were de novo-synthesized in the extract upon addition of circular (+)-PSTVd, were purified by affinity chromatography. This purification avoided contamination by host nucleic acids that had resulted in a misassignment of the start site in an earlier study. Primer-extension analysis of the de novo-synthesized (-)-strands revealed a single start site located in the hairpin loop of the left terminal region in circular PSTVd's secondary structure. This start site is supported further by analysis of the infectivity and replication behavior of site-directed mutants in planta.

Plant Viruses↗
PubMed1992

Base-pair probability profiles of RNA secondary structures.

Dynamic programming algorithms are able to predict optimal and suboptimal secondary structures of RNA. These suboptimal or alternative secondary structures are important for the biological function of RNA. The distribution of secondary structures present in solution is governed by the thermodynamic equilibrium between the different structures. An algorithm is presented which approximates the total partition function by a Boltzmann-weighted summation of optimal and suboptimal secondary structures at several temperatures. A clear representation of the equilibrium distribution of secondary structures is derived from a two-dimensional bonding matrix with base-pairing probability as the third dimension. The temperature dependence of the equilibrium distribution gives the denaturation behavior of the nucleic acid, which may be compared to experimental optical denaturation curves after correction for the hypochromicities of the different base-pairs. Similarly, temperature-induced mobility changes detected in temperature-gradient gel electrophoresis of nucleic acids may be interpreted on the basis of the temperature dependence of the equilibrium distribution. Results are illustrated for natural circular and synthetic linear potato spindle tuber viroid RNA respectively, and are compared to experimental data.

Algorithms↗
PubMed1986

The role of magnesium and potassium ions in the molecular mechanism of ribosome assembly: hydrodynamic, conformational, and thermal stability studies of 16 S RNA from Escherichia coli ribosomes.

In an attempt to understand the role of magnesium ion in ribosome assembly in vitro, the hydrodynamic shape, conformation, and thermal stability of ribosomal 16 S RNA were studied systematically as a function of Mg2+ concentration by sedimentation velocity, intrinsic viscosity, circular dichroism, and difference ultraviolet absorption spectroscopy. These results were then compared with the corresponding parameters obtained for 16 S RNA under the optimal conditions of reconstitution, i.e., at 37 degrees C, 20 mM Mg2+, an ionic strength equal to 0.37, and pH 7.8 [S. H. Allen, and K.-P. Wong (1978) J. Biol. Chem. 253, 8759-8766]. When the 360 mM KCl required for reconstitution of 30 S ribosomes is added to the medium, only subtle conformational changes are observed, consistent with the destabilization of the conformation, thus making the RNA molecule more "open" and accessible to protein binding. However, when the concentration of Mg2+ is lowered from 20 to 1 mM, the hydrodynamic parameters indicate that the 16 S RNA is partially unfolded, while thermal denaturation studies suggest that the amount of base-stacking and base-pairing is not concomitantly altered. Further removal of the Mg2+ by dialysis against a pH 7.8 buffer containing no Mg2+ results in a drastic decrease of secondary structure and indicates that the Mg2+ is required for maintenance of the pairing, stacking, and stability of the nucleotide bases, in addition to the long range interactions which result in a compact structure. The results suggest that the 20 mM Mg2+ is required for the 16 S RNA molecules to assume the proper secondary and tertiary structure containing the protein-binding sites, while the high K+ concentration (360 mM KCl) is needed for "loosening up" the RNA, making the protein binding sites more accessible to the ribosomal proteins for molecular recognition and binding as well as for the conformational changes that occur during ribosome assembly.

Circular Dichroism↗
PubMed1976

Secondary structure of nucleic acids in the folded chromosome from E. coli.

The circular dichroism of membrane-free folded chromosomes from E. coli was measured and analyzed. The spectrum can be explained as a simple linear combination of the individual spectra of E. coli RNA, and E. Coli DNA in the B form. No contribution from A form or C form DNA was detected. There was evidently some real variation in the ratio of the two nucleic acids from preparation to preparation, but the average value was 24% RNA and 76% DNA. No significant light scattering was observed and the analyses indicated no contribution to the circular dichroism from scattering artifacts. Apparently, combining DNA, RNA, and protein into membrane-free folded chromosomes does not change the secondary structure of the DNA or RNA from that found for the free nucleic acid in the same solvent system.

Chromosomes, Bacterial↗
PubMed1988

Nucleotide sequence predicts circularity and self-cleavage of 300-ribonucleotide satellite of arabis mosaic virus.

The nucleotide sequence of the satellite of arabis mosaic virus was determined using the satellite RNA encapsidated in virions. The 300-nucleotide long sequence showed extensive homology (50%) with that of the 359-nucleotide satellite RNA of tobacco ringspot virus, which occurs both in a linear and a circular form. This homology also revealed the presence of conceived sequences believed to mediate self-cleavage of the latter as well as other viral satellite RNAs. A circular form of the arabis mosaic virus satellite can be isolated from infected tissues and partially converts to the linear form upon elution from denaturing gels.

Base Sequence↗
PubMed2006

RNA binding small molecules: studies on t-RNA binding by cytotoxic plant alkaloids berberine, palmatine and the comparison to ethidium.

The interaction of two natural protoberberine plant alkaloids berberine and palmatine with t-RNA(phe) was studied using various biophysical techniques and the data was compared with the binding of the classical DNA intercalator, ethidium. The results of optical thermal melting, differential scanning calorimetry and circular dichroism characterized the native cloverleaf structure of t-RNA under the conditions of the study. The strong binding of the alkaloids and ethidium to t-RNA was revealed from the absorption and fluorescence studies. The salt dependence of the binding constants enabled the dissection of the binding free energy to electrostatic and non-electrostatic contributions. This analysis revealed a surprisingly large favourable component of the non-electrostatic contribution to the binding of these charged alkaloids and ethidium to t-RNA. Isothermal titration calorimetric studies revealed that the binding of both the alkaloids is driven by a moderately favourable enthalpy decrease and a moderately favourable entropy increase while that of ethidium is driven by a large favourable enthalpy decrease. Taken together, the results suggest that the binding of these alkaloid molecules on the t-RNA structure appears to be mostly by partial intercalation while ethidium intercalates to the t-RNA. These results reveal the molecular aspects on the interaction of these alkaloids to t-RNA.

Alkaloids↗
PubMed1999

Spectroscopic studies on the interaction of aristololactam-beta-D-glucoside with DNA and RNA double and triple helices: A comparative study.

The interaction of aristololactam-beta-D-glucoside (ADG), a DNA intercalating alkaloid, with the DNA triplexes, poly(dT). poly(dA)xpoly(dT) and poly(dC).poly(dG)xpoly(dC+), and the RNA triplex poly(rU).poly(rA)xpoly(rU) was investigated by circular dichroic, UV melting profile, spectrophotometric, and spectrofluorimetric techniques. Comparative interaction with the corresponding Watson-Crick duplexes has also been examined under identical experimental conditions. Triplex formation has been confirmed from biphasic thermal melting profiles and analysis of temperature-dependent circular dichroic measurements. The binding of ADG to triplexes and duplexes is characterized by the typical hypochromic and bathochromic effects in the absorption spectrum, quenching of steady-state fluorescence intensity, a decrease in fluorescence quantum yield, an increase or decrease of thermal melting temperatures, and perturbation in the circular dichroic spectrum. Scatchard analysis indicates that ADG binds both to the triplexes and the duplexes in a noncooperative manner. Binding parameters obtained from spectrophotometric measurements are best fit by the neighbor exclusion model. The binding affinity of ADG to the DNA triplexes is substantially stronger than to the RNA triplex. Thermal melting study further indicates that ADG stabilizes the Hoogsteen base-paired third strand of the DNA triplexes whereas it destabilizes the same strand of RNA triplex but stabilizes its Watson-Crick strands. Comparative data reveal that ADG exhibits a stronger binding to the triple helical structures than to the respective double helical structures.

Alkaloids↗