Structure of EDTA-treated satellite tobacco necrosis virus at pH 6.5

The crystal structure of EDTA-treated satellite tobacco necrosis virus (STNV) at pH 6.5 has been determined to 7.5 A resolution (1 A = 0.1 nm) with molecular replacement techniques, using the known structure of the protein subunit. The calcium ions at the 3-fold contacts are absent, whereas the calcium ions on the 5-fold axes still remain. The protein shell is slightly expanded. The expansion does not impose any large conformational changes on the subunits and the subunit contacts are to a large extent retained. The electron density map shows high levels of density in the RNA region. It is found close to the protein shell but well-separated from the protein. This density indicates a preferential ordering of the RNA in certain regions, but does not allow a detailed interpretation of the RNA conformation. A similar density in the RNA region is also found in a low resolution map of native STNV.     

Vault nanocapsule dissociation into halves triggered at low pH

Vaults are self-assembled ribonucleoprotein nanocapsules that consist of multiple copies of three proteins (major vault protein, VPARP, and TEP1) and an untranslated RNA. Although their function has not been determined, vaults are found in nearly all eukaryotic cells. This study describes the use of fluorescence spectroscopy, multiangle laser light scattering (MALLS), and the quartz crystal microbalance (QCM) as tools in investigating recombinant vault conformational change in response to a varied solution pH. Identification of conditions for reversible vault disassembly and reassembly could enable application of these nanocapsules in drug delivery and in nanomaterials synthesis. Initial monitoring of changes in the intrinsic fluorescence intensity of vaults showed a 60% increase at pH 3.4 compared to that at pH 6.5, suggesting vaults exhibit a more open conformation at low pH. Fluorescence quenching studies provided further evidence of a vault structural change at low pH. MALLS data suggested a decrease in molecular mass accompanied by a clear increase in the radius of gyration as the solution pH was shifted from 6.5 to 3.4. This result prompted the hypothesis that vaults dissociate at least partially at low pH. Using the QCM to study adsorption of the vault onto self-assembled monolayers, data that suggest vault dissociation at low pH, even when the vault is in an adsorbed state, were also obtained. Finally, transmission electron microscopy (TEM) of negatively stained vaults at pH 6.5 and 3.4 confirmed the fluorescence spectroscopy, MALLS, and QCM findings by providing visual evidence that vaults disassemble into halves as the solution pH is lowered from 6.5 to 3.4.     

Cultivation of Endomyces magnusii yeasts under batch and continuous conditions at an acid pH

The energy parameters of Endomyces magnusii cells and mitochondria were studied under the conditions of batch and continuous cultivation at different pH of the medium containing ethanol. The yeast was found to be capable of growth in the chemostat regime at D=0.2 h-1. Changes in the pH of the medium from 3.0 to 5.6 almost did not change the parameters characterizing oxidative phoshorylation of the mitochondria (the respiration chain contained three phosphorylation points). This correlated with the nearly identical biomass yield and economical coefficient. The content of RNA, DNA and protein remained unchanged at different pH values whereas the content of lipids increased at acid pH.     

Differentiation of Nucleic Acids by Staining at Controlled pH and by A Schiff-Methylene Blue Sequence

Fixation with Bouin's fluid preserves cytoplasmic and nucleolar ribonucleic acid (UNA) particularly well. RNA may be demonstrated preferentially in Bouin fixed tissue by staining with 0.02% thiazine dye in aqueous McIIvaine phosphate-citrate buffer between pH 3 and 4. Methylation blockage of basophilia other than that of nucleic acids permits staining of RNA with thiazine dyes near neutrality. The deoxyribonucleic acid (DNA) of chromatin undergoes a Feulgen type hydrolysis in the tissue block during 24 hr fixation with Bouin's fluid. This hydrolysis by picric acid permits Schiff staining of the DNA wthout further acid hydrolysis. Consequently after Bouin fixation it is possible to demonstrate DNA and RNA specifically by a Schiff-methylene blue sequence. Thus a Schiff stain without further acid hydrolysis followed by 0.02% methylene blue in phosphate-citrate buffer at pH 3.0 to 3.5 colors DNA magenta in contrast to the blue of RNA.     

Binding of oligonucleotides to cell membranes at acidic pH.

Antisense oligodeoxynucleotides [oligo(dN)] have the ability to enter living cells and block the expression of specific genes. However, little is known about the mechanism of cellular uptake of oligo(dN). We have found that oligo(dN) can bind to the cell membranes of eukaryotic cells with much greater efficiency under acidic conditions (pH 4.0-4.5) than at neutral pH. The binding appears to be specific to poly nucleic acids since various sizes of oligo(dN), DNA and RNA, but not mononucleotides, compete for the binding. We have identified a 34 kDa membrane protein from T-cells, which binds to oligo(dT) cellulose at pH 4.5 and can be eluted at pH 7.5. This protein fraction blocked the binding of oligo(dN) to living T-cells in a competitive fashion. Our results suggest that eukaryotic cells have a receptor for oligo(dN) at acidic pH and that the 34 kDa dalton protein on the cell membrane may mediate such binding.     

Wheat embryo ribonucleates. XII. Formal characterization of terminal and penultimate nucleoside residues at the 5'-ends of "capped" RNA from imbibing wheat embryos

(1) N2,N2,7-Trimethylguanosine, not previously detected as a component of plant RNA, is shown to be present in the RNA which is isotopically labelled when dry wheat embryos imbibe water in a medium that contains[methyl-3H]methionine. (2) N2,N2,7-Trimethylguanosine and 7-methylguanosine are released as part of "capped" oligonucleotides when the isotopically labelled RNA from imbibing wheat embryos is subjected to hydrolysis by RNase T2. (3) By way of contrast with the "capped" RNA of animal cells, 5'-terminal "cap" structures (m7Gppp- and m32,2,7 Gppp-) in the "capped" RNA from the higher plant organism are not bonded to pneultimate O2'-methylnucleoside constituents. (4) In an allied study, it has been found that recovery of poly (A)-rich RNA from dry wheat embryos depends on the inclusion of sodium dodecyl sulphate (SDS) in phosphate-buffered (pH 6.8) phenolic emulsions. By way of contrast, recovery of poly (A)-rich RNA from dry wheat embryos does not depend on the inclusion of SDS in Tris (hydroxymethyl) aminomethane buffered (pH 9.0) phenolic emulsions.     

Enhanced nucleic acid binding to ATP-bound hepatitis C virus NS3 helicase at low pH activates RNA unwinding

The molecular basis of the low-pH activation of the helicase encoded by the hepatitis C virus (HCV) was examined using either a full-length NS3 protein/NS4A cofactor complex or truncated NS3 proteins lacking the protease domain, which were isolated from three different viral genotypes. All proteins unwound RNA and DNA best at pH 6.5, which demonstrate that conserved NS3 helicase domain amino acids are responsible for low-pH enzyme activation. DNA unwinding was less sensitive to pH changes than RNA unwinding. Both the turnover rate of ATP hydrolysis and the K_m of ATP were similar between pH 6 and 10, but the concentration of nucleic acid needed to stimulate ATP hydrolysis decreased almost 50-fold when the pH was lowered from 7.5 to 6.5. In direct-binding experiments, HCV helicase bound DNA weakly at high pH only in the presence of the non-hydrolyzable ATP analog, ADP(BeF₃). These data suggest that a low-pH environment might be required for efficient HCV RNA translation or replication, and support a model in which an acidic residue rotates toward the RNA backbone upon ATP binding repelling nucleic acid from the binding cleft.     

Circular dichroism studies of the HIV-1 Rev protein and its specific RNA binding site

The circular dichroism (CD) spectrum of the Rev protein from HIV-1 indicates that Rev contains about 50% alpha helix and 25% beta sheet at 5 degrees C in potassium phosphate buffer, pH 3, and 300 mM KF. The spectrum is independent of protein concentration over a 20-fold range. At neutral pH, Rev is relatively insoluble but can be brought into solution by binding to its specific RNA binding site, the Rev-responsive element (RRE), at a Rev:RNA ratio of about 3:1. Nonspecific binding to tRNA does not solubilize Rev. As judged by difference CD spectra, the conformation of Rev when bound to the RRE at neutral pH is similar to the conformation of unbound Rev at pH 3, although changes in the RNA may also contribute to the difference spectrum. Indeed, some difference is observed near 260 nm, consistent with a conformational change of the RRE upon Rev binding. Rev alone at pH 3 shows irreversible aggregation as the temperature is raised, while Rev bound to the RRE at neutral pH shows a reversible transition with a Tm of 68 degrees C.     

Activity of glutaraldehyde at low concentrations (less than 2%) against poliovirus and its relevance to gastrointestinal endoscope disinfection procedures.

The activity of glutaraldehyde (GTA) at low concentrations (less than 2%) against poliovirus was assessed by a suspension procedure. The inactivation kinetics showed that concentrations of less than or equal to 0.10% were effective against purified poliovirus at pH 7.2; a 1 log10 reduction was obtained in 70 min with 0.02% GTA, and a 3 log10 reduction was obtained in 30 min with 0.10% GTA. GTA activity at low concentrations was greatly enhanced at alkaline pH, but was completely abolished at acid pH. In contrast, the inactivation assays on poliovirus RNA showed that it was highly resistant to GTA at concentrations up to 1.0% at pH 7.2. At pH 8.3 a low inactivation was noticed with 1.0% GTA. Our results are of relevance to hospital practice in digestive endoscopy investigations because there has been an increasing tendency to use low concentrations of GTA and very short contact times in disinfection procedures.     

Activity of glutaraldehyde at low concentrations (less than 2%) against poliovirus and its relevance to gastrointestinal endoscope disinfection procedures.

The activity of glutaraldehyde (GTA) at low concentrations (less than 2%) against poliovirus was assessed by a suspension procedure. The inactivation kinetics showed that concentrations of less than or equal to 0.10% were effective against purified poliovirus at pH 7.2; a 1 log10 reduction was obtained in 70 min with 0.02% GTA, and a 3 log10 reduction was obtained in 30 min with 0.10% GTA. GTA activity at low concentrations was greatly enhanced at alkaline pH, but was completely abolished at acid pH. In contrast, the inactivation assays on poliovirus RNA showed that it was highly resistant to GTA at concentrations up to 1.0% at pH 7.2. At pH 8.3 a low inactivation was noticed with 1.0% GTA. Our results are of relevance to hospital practice in digestive endoscopy investigations because there has been an increasing tendency to use low concentrations of GTA and very short contact times in disinfection procedures.     

Design and NMR analysis of HDV ribozymes for structural investigation.

Three variants of minimized hepatitis delta virus (HDV) RNA ribozyme systems (Rz-1 to approximately Rz-3) (Fig. 1) were designed on the basis of the "pseudoknot" structure model and synthesized. Rz-1 is a cis-acting ribozyme system (a cleaved form, 56-mer) in which stem IV is deleted from the active domain of genomic HDV RNA. Rz-1 was uniformly labeled with stable isotopes, 13C and 15N. The 2D-NOESY and 2D-HSQC data for Rz-1 suggest that Rz-1 forms the pseudoknot structure and G38 which is opposite to the cleavage site makes a base-pair. Rz-2 is a trans-acting ribozyme system which consists of three RNA oligomer strands (substrate: 8-mer, the cytidine residue at the cleavage site is replaced by 2'-O-methylcytidine; enzyme: 16-mer plus 35-mer). Rz-3 is a ribozyme in which the three RNA strands of Rz-2 are connected. It turns out that Rz-3 forms an inactive structure with low cleavage activity (k(obs) = 0.009) and final cleavage yield (6%). Rz-3 has the highest cleavage activity at pH 5.5. The optimal activity at acidic pH is similar to that of the wild type ribozyme. We also synthesized and examined the activity and structure of Rz-4 (designed by Perrotta and Been) which consists of two RNA strands (1).     

Imino proton exchange in the 5S RNA of Escherichia coli and its complex with protein L25 at 490 MHz

Imino proton exchange has been examined by NMR in the 5S RNA of Escherichia coli, its principal RNase A resistant fragment, fragment 1 (bases 1-11, 69-120), and complexes between that fragment and ribosomal protein L25 by using both real-time and relaxation techniques. Fragment 1 RNA imino protons exchange at rates between 0.5 and 15 s-1 at 303 K in 5 mM cacodylate buffer, pH 7.4. In contrast with many tRNAs, intact 5S RNA contains no imino protons with exchange lifetimes as great as 1 min. Consistent with the results of Gueron and his colleagues [Leroy, J. L., Bolo, N., Figueroa, N., Plateau, P., & Gueron, M. (1985) J. Biomol. Struct. Dyn. 2,915-939; Leroy, J. L., Broseta, D., & Gueron, M. (1985) J. Mol. Biol. 184, 165-178] with tRNA, exchange in 5S RNA is catalyst-limited under conditions generally used for imino proton spectroscopy, such as those given above. Using Gueron's catalyst saturation technique, base pair opening rates have been measured for several AU and GU base pairs in fragment 1. They range from 50 to 300 s-1 at 303 K and depend on base pair type and also to some degree on context. Similar studies have been done on complexes of L25 and fragment 1. The binding of L25 to fragment 1 reduces the exchange rate of many imino protons within the region to which it binds, consistent with the hypothesis that its binding stabilizes the secondary structure of 5S RNA.     

Characteristics of pulse-labeled ribonucleic acid in relation to medium pH and acetate concentration during meiosis of yeast

Summary Cellular impermeability associated with sporulating cells of Saccharomyces cerevisiae is caused by a rapid increase in the medium pH. Three factors have been identified as being important in regulating the rise in medium pH: 1) the cell density, 2) the potassium acetate concentration of the sporulation medium, and 3) and initial pH below 6.0. Sporulation conditions were established for strain 4579 which resulted in optimum uptake of ³H-adenine at T₇, a period when the cells would be normally impermeable. Pulse-labeled polysomal RNA was characterized at T₄ in naturally permeable cells of strain SK-1 and impermeable cells which required manipulation of the medium pH to facilitate uptake. Transfer ribonucleic acid (RNA), poly A-containing RNA and ribosomal RNA were synthesized in both cultures during the 20 min pulse. Furthermore, the rate of ribosomal RNA synthesis and processing into functional ribosomes approached the rate reported for vegetative cells. Initial sporulation conditions which caused a prolonged delay in the rise in medium pH adversely affected the kinetics of appearance and number of ascospores. The affect was shown to be on meiotic events since a reduction of sporulation was always accompanied by a reduction in the amount of intragenic recombination.     

Polyadenylated RNA from Vicia faba meristematic root cells. Localization and size estimation of the poly (A) segment.

After incubating root apices from two-day-old bean seedlings with [3H] adenine the RNA was extracted from whole cells or polysomes, and the poly (A) sequences were isolated by nuclease digestion followed by poly(U)-Sepharose chromatography. The alterations of the RNA molecules due to the various treatments were monitored by sucrose density gradients. It was found that sequential extraction first at pH 7.6 then at pH 9.0 did not result in a separation between RNA poor in poly(A) sequences and poly(A)-rich RNA. Furthermore chromatography analysis of hydrolysates from nuclease-resistant RNA extracted either at pH 7.6 or pH 9.0 revealed that AMP constituted nearly 95% of the bases and that the poly(A) sequences, about 200 bases, were located at the 3' terminus of the polyadenylated RNA. No size difference was found for the poly(A) segment between the pH-7.6-extracted RNA and that extracted at pH 9.0.     

Ribonucleic acid from coffee beans

A method of extraction of RNA from coffee based on phenol treatment is described. Effectsf of various agents and pH of the extracting buffer on the efficiency of extraction were studied. The best extracting solution is 0·2 M Tris-HCl buffer at pH 7·4 with 1% sodium dodecyl sulphate and 0·05% EDTA. RNA (5–6%) is lost in the tissue residue and 4·6% in the interphase layer. No significant deviation of the spectral characteristics of the RNA solutions obtained from three samples of coffee from that for purified yeast RNA is observed. The purine-pyrimidine ratio for the RNA has been found to be in the range of 1·25–1·38.