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At least 1,063 records · Page 59Linked to original sources
PubMed1978

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.

Animals↗
PubMed1993

Raman dynamic probe of hydrogen exchange in bean pod mottle virus: base-specific retardation of exchange in packaged ssRNA.

We describe a novel approach to investigating exchange kinetics in biological assemblies. The method makes use of a Raman multichannel analyzer coupled with a dialysis flow cell. We employ this methodology to determine exchange rates of labile hydrogens in both the packaged RNA genome and protein subunits of bean pod mottle virus (BPMV). In the BPMV assembly, which is similar to human picornaviruses, the x-ray structure indicates that about 20% of the ssRNA chain is ordered at the threefold vertices of the icosahedral capsid, although the nucleotide bases in the ordered segments are not known (Chen et al., 1989). Here, we compare exchange profiles of the native virus with those of the empty capsid, model nucleic acids and aqueous solvent to reveal the following exchange characteristics of BPMV RNA and protein: (i) Base-specific retardation of exchange is observed in the packaged RNA. (ii) Retardation is greatest for uracil residues, for which the first-order exchange rate constant (kU = 0.18 +/- 0.02 min-1) is 40% lower than that of either the H2O solvent or adenine or cytosine groups of RNA (ksolv approximately kA approximately kC = 0.30 +/- 0.02 min-1). (iii) Retardation of exchange is also observed for the guanine residues of packaged RNA. (iv) No appreciable exchange of amide NH groups of capsid subunits occurs within the time of complete exchange (t approximately 10 min) of packaged RNA or bulk solvent. Thus, the present results identify sites in both the protein subunits (amide NH) and RNA nucleotides (amino NH2 and imino NH) which are resistant to solvent-catalyzed hydrogen exchange. We propose that retardation of exchange of labile sites of the RNA nucleotides is a consequence of the organization of the RNA chromosome within the virion. Our findings support a model for BPMV in which surface and buried domains of capsid subunits are extensively and rigidly hydrogen-bonded, and in which uracil and guanine exocyclic donor groups of packaged RNA are the principal targets for subunit interaction at the threefold vertices of the capsid.

Biophysical Phenomena↗
PubMed1997

Nucleic acid vibrational circular dichroism, absorption, and linear dichroism spectra. I. A DeVoe theory approach.

Infrared (IR) vibrational circular dichroism (VCD), absorption, and linear dichroism (LD) spectra of four homopolyribonucleotides, poly(rA), poly(rG), poly(rC), and poly(rU), have been calculated, in the 1750-1550 cm-1 spectral region, using the DeVoe polarizability theory. A newly derived algorithm, which approximates the Hilbert transform of imaginaries to reals, was used in the calculations to obtain real parts of oscillator polarizabilities associated with each normal mode. The calculated spectra of the polynucleotides were compared with previously measured solution spectra. The good agreement between calculated and measured polynucleotide spectra indicates, for the first time, that the DeVoe theory is a useful means of calculating the VCD and IR absorption spectra of polynucleotides. For the first time, calculated DeVoe theory VCD and IR absorption spectra of oriented polynucleotides are presented. The calculated VCD spectra for the oriented polynucleotides are used to predict the spectra for such measurements made in the future. The calculated IR spectra for the oriented polynucleotides are useful in interpreting the linear dichroism of the polynucleotides.

Circular Dichroism↗
PubMed1982

Polycytidylate hydrolysing ribonuclease variants of human pancreas.

Human pancreatic ribonuclease (RNase) comprises three variants with isoelectric points at pH 4.8, pH 9.3 and pH 9.7. These variants are not artifacts of the purification procedure, since they can be demonstrated in crude pancreatic extracts obtained with mild procedures (4). Although these RNase variants differ in their isoelectric points, they are similar in all the other properties so far studied. With polycytidylic acid as a substrate, their activities are optimal in 0.05M sodium phosphate buffer at pH 6.5. Of the several buffers tested, sodium phosphate buffer yielded maximal enzyme activity, but the concentration of phosphate is highly critical. Borate concentrations of 0.05M and 0.1 M respectively brought about 25% and 50% reductions in enzyme activity. Presence of phosphate in the reaction mixture obviates to a great extent the borate inhibition. These RNase variants are highly specific for the secondary phosphate esters of cytidine 3'-phosphate. They have no measurable activity on polyadenylic and polyguanylic acids and their activity on polyuridylic acid is less than 2% of that on polycytidylic acid. These RNase variants could serve as distinctive biochemical markers for the pancreas.

Borates↗
PubMed2003

The ubiquitin-protein ligase activity of Hdm2 is inhibited by nucleic acids.

The proto-oncoprotein Hdm2 is a member of the RING finger-type family of ubiquitin-protein ligases E3. The RING finger domain is assumed to mediate the specific interaction of an E3 with its cognate ubiquitin-conjugating enzyme E2, which catalyzes the covalent attachment of ubiquitin to substrate proteins. In addition, the RING finger domain of Hdm2 is involved in Hdm2 homooligomer formation and has the capacity to bind to RNA in a sequence-specific manner. Here we report that interaction with nucleic acids interferes with both Hdm2/Hdm2 complex formation and auto-ubiquitination of Hdm2 in vitro. Furthermore, although binding of Hdm2 to the tumor suppressor p53 is not inhibited by nucleic acids, Hdm2-mediated ubiquitination of p53 is significantly decreased. Taken together, these results provide the first example of an E3 whose activity can be regulated by direct interaction with nucleic acids.

Base Sequence↗
PubMed1985

Neonucleoproteins. Preparation and high-performance liquid chromatographic characterization of succinyl-lysozyme-diaminooctyl-polycytidylic acid and related polycytidylic acid conjugates.

Transamination conjugates of cytidine-3'-phosphate and polycytidylic acid (poly C) with diaminopropane, diaminohexane and diaminooctane (DAO) are formed both at 25 degrees C and 60 degrees C. The extent of reaction and formation of side products, with intermittent hydrolysis to mononucleotides in the case of aminoalkyl-poly C, is monitored by reversed-phase high-performance liquid chromatography. Both Dns-DAO-poly C and succinyl-lysozyme-DAO-poly C covalent conjugates are then prepared and similarly characterized, including separation on a size-exclusion diol high-performance liquid chromatography column. The retention of the latter on a wide-pore reversed-phase column seems to be controlled by the protein moiety.

Chemical Phenomena↗
PubMed2002

Mutational analysis of polynucleotide phosphorylase from Escherichia coli.

Polynucleotide phosphorylase (PNPase), a homotrimeric exoribonuclease present in bacteria, is involved in mRNA degradation. In Escherichia coli, expression of this enzyme is autocontrolled at the translational level. We introduced about 30 mutations in the pnp gene by site-directed mutagenesis, most of them in phylogenetically conserved residues, and determined their effects on the three catalytic activities of PNPase, phosphorolysis, polymerisation and phosphate exchange, as well as on the efficiency of translational repression. The data are presented and discussed in the light of the crystallographic structure of PNPase from Streptomyces antibioticus. The results show that both PNPase activity and the presence of the KH and S1 RNA-binding domains are required for autocontrol. Deletions of these RNA-binding domains do not abolish any of the three catalytic activities, indicating that they are contained in a domain independent of the catalytic centre. Moreover, the catalytic centre was located around the tungsten-binding site identified by crystallography. Some mutations affect the three catalytic activities differently, an observation consistent with the presence of different subsites.

Amino Acid Sequence↗
PubMed2002

Protein-protein interactions between human exosome components support the assembly of RNase PH-type subunits into a six-membered PNPase-like ring.

The exosome is a complex of 3'-->5' exoribonucleases, which functions in a variety of cellular processes, all requiring the processing or degradation of RNA. Here we present a model for the assembly of the six human RNase PH-like exosome subunits into a hexameric ring structure. In part, this structure is on the basis of the evolutionarily related bacterial degradosome, the core of which consists of three copies of the PNPase protein, each containing two RNase PH domains. In our model three additional exosome subunits, which contain S1 RNA-binding domains, are positioned on the outer surface of this ring. Evidence for this model was obtained by the identification of protein-protein interactions between individual exosome subunits in a mammalian two-hybrid system. In addition, the results of co-immunoprecipitation assays indicate that at least two copies of hRrp4p and hRrp41p are associated with a single exosome, suggesting that at least two of these ring structures are present in this complex. Finally, the identification of a human gene encoding the putative human counterpart of the bacterial PNPase protein is described, which suggests that the exosome is not the eukaryotic equivalent of the bacterial degradosome, although they do share similar functional activities.

Amino Acid Sequence↗
PubMed2003

Human polynucleotide phosphorylase, hPNPase, is localized in mitochondria.

The human gene encoding a polynucleotide phosphorylase (hPNPase) has been recently identified as strongly up-regulated in two processes leading to irreversible arrest of cell division: progeroid senescence and terminal differentiation. Here, we demonstrate that the hPNPase is localized in mitochondria. Our finding suggests the involvement of mitochondrial RNA metabolism in cellular senescence.

Cloning, Molecular↗