The Mechanical Properties of RNA-DNA Hybrid Duplex Stretched by Magnetic Tweezers

RNA can anneal to its DNA template to generate an RNA-DNA hybrid (RDH) duplex and a displaced DNA strand, termed R-loop. RDH duplex occupies up to 5% of the mammalian genome and plays important roles in many biological processes. The functions of RDH duplex are affected by its mechanical properties, including the elasticity and the conformation transitions. The mechanical properties of RDH duplex, however, are still unclear. In this work, we studied the mechanical properties of RDH duplex using magnetic tweezers in comparison with those of DNA and RNA duplexes with the same sequences. We report that the contour length of RDH duplex is ～0.30 nm/bp, and the stretching modulus of RDH duplex is ～660 pN, neither of which is sensitive to NaCl concentration. The persistence length of RDH duplex depends on NaCl concentration, decreasing from ～63 nm at 1 mM NaCl to ～49 nm at 500 mM NaCl. Under high tension of ～60 pN, the end-opened RDH duplex undergoes two distinct overstretching transitions; at high salt in which the basepairs are stable, it undergoes the nonhysteretic transition, leading to a basepaired elongated structure, whereas at low salt, it undergoes a hysteretic peeling transition, leading to the single-stranded DNA strand under force and the single-stranded RNA strand coils. The peeled RDH is difficult to reanneal back to the duplex conformation, which may be due to the secondary structures formed in the coiled single-stranded RNA strand. These results help us understand the full picture of the structures and mechanical properties of nucleic acid duplexes in solution and provide a baseline for studying the interaction of RDH with proteins at the single-molecule level.     

Fractionation of protein, RNA, and plasmid DNA in centrifugal precipitation chromatography using cationic surfactant CTAB containing inorganic salts NaCl and NH(4)Cl

Centrifugal precipitation chromatography (CPC) is a separation system that mainly employs a moving concentration gradient of precipitating agent along a channel and solutes of interest undergo repetitive precipitation-dissolution, fractionate at different locations, and elute out from the channel according to their solubility in the precipitating agent solution. We report here for the first time the use of a CPC system for fractionation of protein, RNA, and plasmid DNA in clarified lysate produced from bacterial culture. The cationic surfactant cetyltrimethylammonium bromide (CTAB) was initially used as a precipitating agent; however, all biomolecules showed no differential solubility in the moving concentration gradient of this surfactant and, as a result, no separation of protein, RNA, and plasmid DNA occurred. To overcome this problem, inorganic salts such as NaCl and NH(4)Cl were introduced into solution of CTAB. The protein and RNA were found to have higher solubility with the addition of these salts and separated from the plasmid DNA. Decreasing surface charge density of CTAB upon addition of NaCl and NH(4)Cl was believed to lead to lower surfactant complexation, and therefore caused differential solubility and fractionation of these biomolecules. Addition of CaCl(2) did not improve solubility and separation of RNA from plasmid DNA.     

Salt induced conformational transition between A and Z forms of r(CGCGCG) as revealed by a Raman spectroscopic study

Solution conformation in different conditions of r(CGCGCG) has been studied by a Raman spectroscopic method. In NaCl solution, r (CGCGCG) takes only an A-form duplex in which guanosine and cytidine have C3'endo-anti conformation even at 5M salt concentration. In much higher ionic strength condition (5M NaCl plus 1M MgCl2 or 6M NaClO4), it undergoes a transition to a left-handed Z-form. The Raman spectrum of the Z-form RNA was found to be very similar to that of Z-form DNA, suggesting that Z-RNA involves a C3'endo-syn guanosine and an in between form of C2'endo-Cl'exo-anti cytidine.     

Formation of stable nanoparticles via electrostatic complexation between sodium caseinate and gum arabic

The formation of electrostatic complexes between sodium caseinate and gum arabic (GA) was studied as a function of pH (2.0-7.0), using slow acidification in situ with glucono-delta-lactone (GDL) or titration with HCl. The colloidal behavior of the complexes under specific conditions was investigated using absorbance measurements (at 515 or 810 nm) and dynamic light scattering (DLS). In contrast to the sudden increase in absorbance and subsequent precipitation of sodium caseinate solutions at pH < 5.4, the absorbance values of mixtures of sodium caseinate and GA increased to a level that was dependent on GA concentration at pH 5.4 (pH(c)). The absorbance values remained constant with further decreases in pH until a sudden increase in absorbance was observed (at pH(phi)). The pH(phi) was also dependent upon the GA concentration. Dynamic light scattering (DLS) data showed that the sizes of the particles formed by the complexation of sodium caseinate and GA between pH(c) and pH(phi) were between 100 and 150 nm and these nanoparticles were visualized using negative staining transmission electron microscopy (TEM). Below pH(phi), the nanoparticles associated to form larger particles, causing phase separation. zeta-Potential measurements of the nanoparticles and chemical analysis after phase separation showed that phase separation was a consequence of charge neutralization. The formation of complexes between sodium caseinate and GA was inhibited at high ionic strength (>50 mM NaCl). It is postulated that the structure of the nanoparticles comprises an aggregated caseinate core, protected from further aggregation by steric repulsion of one, or more, electrostatically attached GA molecules.     

Self-association equilibria of Escherichia coli UvrD helicase studied by analytical ultracentrifugation

The Escherichia coli UvrD protein (helicase II) is an SF1 superfamily helicase required for methyl-directed mismatch repair and nucleotide excision repair of DNA. We have characterized quantitatively the self-assembly equilibria of the UvrD protein as a function of [NaCl], [glycerol], and temperature (5-35 degrees C; pH 8.3) using analytical sedimentation velocity and equilibrium techniques, and find that UvrD self-associates into dimeric and tetrameric species over a range of solution conditions (t相似文献   

UV-induced pyrimidine dimers and trimethylpsoralen cross-links do not alter chromatin folding in vitro

We have examined the ability of intact and histone H1 depleted chromatin fibers to fold into higher ordered structures in vitro following DNA damage by two different agents: UV irradiation at 254 nm and trimethylpsoralen plus near-UV light. Both agents damage DNA specifically, yet cause different degrees of unwinding (and possibly bending) of the DNA helix. In addition, trimethylpsoralen forms interstrand DNA cross-links. The structural transitions of intact and histone H1 depleted chromatin fibers, induced by NaCl, were monitored by analytical ultracentrifugation, light scattering, and circular dichroism. Our results indicate that when chromatin fibers contain even large, nonphysiological amounts of DNA photodamage by either agent, the salt-induced folding of these fibers into higher ordered structures is unaffected. The compact 30-nm fiber must therefore be able to accommodate a large amount of DNA photodamage (greater than one UV-induced photoproduct or trimethylpsoralen interstrand cross-link per nucleosome) with little or no change in the overall size or compaction of this structure.     

Reaction rate of OH radicals with phi X174 DNA: influence of salt and scavenger

A derivation is given for the dependence of the rate constant of the reaction of OH radicals with a spherical macromolecule on the rate by which such radicals are scavenged by the medium. Experiments were carried out with oxygenated solutions of dilute single-stranded phi X174 DNA at 10(-4)M NaCl (large reaction radius of DNA) or at 10(-4)M NaCl + MgCl2 (small reaction radius) with t-butanol as a scavenger. The results of these experiments cannot be described by simple second-order competition, but can be explained by the predicted dependence of the rate constant of the reaction OH + DNA on the concentration of t-butanol. Furthermore, the results show that only part of the reactions of OH radicals with phi X174 DNA leads to DNA inactivation, and that even at zero scavenger concentration OH radicals are scavenged by other molecules than DNA, presumably impurities remaining even after careful purification of the DNA.     

DNA,RNA, and protein synthesis in the cyanobacteriumSynechocystis sp. PCC 6803 adapted to different salt concentrations

A salt shock of 684mm NaCl reduced RNA and DNA synthesis to about 30% of the control level inSynechocystis. DNA synthesis recovered to the initial level within 4 h, while for recovery of RNA synthesis about 8 h were necessary. In cells completely adapted to different salt concentrations (from 171 to 1026mm NaCl), a continuous decrease in the RNA content with increasing salt concentrations up to 684mm NaCl was found, whereas the lowest DNA content was measured around 342mm NaCl, i.e., the salinity at which maximal growth occurred. With the uracil and thymidien incorporation technique, maxima in DNA and RNA synthesis were detected in control cells. Comparing these rates with nucleic acid synthesis rates calculated from the contents of DNA and RNA and the growth rates indicated that adaptation to 1026mm NaCl seemed to lead to an increased RNA turnover inSynechocystis. Analysis of protein synthesis with³⁵S-methionine labeling showed alterations in salt-adapated cells ofSynechocystis. At least three proteins (20.5, 25.8, and 35.8 kDa) were synthesized with highest rates at salinities leading to maximal growth, the synthesis of nine proteins (12.5, 16.9, 19.2, 22.2, 24.7, 28.5, 30.5, 50.3, and 63.5 kDa) increased and that of several other proteins decreased with increasing salinity; but only three proteins (12.5, 22.2, and 30.5 kDa) accumulated under these conditions. The adaptation ofSynechocystis to enhanced salt concentrations led also to increased contents of glucosylglycerol, glycogen, and significant amounts of K⁺ as well as Na⁺ ions.     

Similarity of hnRNA sequences in blastula and pluteus stage sea urchin embryos

We have compared the total single-copy sequences transcribed as nuclear RNA in blastula and pluteus stage embryos of the sea urchin Tripneustes gratilla by hybridization of excess nuclear RNA with purified radioactive single-copy DNA. The kinetics of hybridization of either blastula or pluteus nuclear RNA with single-copy DNA show a single pseudo-first-order reaction with 34% of the single-copy genome. From the rate of the reaction and the purity of the nuclear RNA, it can be estimated that the reacting RNAs are present on the average at a concentration of one molecule per 14 nuclei. A mixture of blastula and pluteus RNA also hybridizes with 34% of the single-copy genome, indicating that the total complexity of RNAs transcribed at both stages is no greater than transcribed at each stage alone. The identity of the sequences transcribed by blastula and pluteus embryos was further examined by fractionation of the labeled DNA into sequences complementary and not complementary to pluteus RNA. This was achieved by hybridization of single-copy DNA to high pluteus RNA Cot, and separation of the hybridized and nonhybridized DNA on hydroxylapatite. Using either the DNA complementary or noncomplementary with pluteus RNA, essentially identical amounts of RNA:DNA hybrids are formed at high RNA Cot with blastula or pluteus RNA. Gross changes in the total RNA sequences transcribed do not appear to be involved in the developmental changes between blastula and pluteus, even though 45% of the mRNA sequences change between these two stages (Galau et al., 1976).     

Superhelical DNA studied by solution scattering and computer models

We present here recent results on the structure of superhelical DNA and its changes with salt concentration between 0.01 and 1.5 M NaCl. Scattering curves of two different superhelical DNAs were determined by static light scattering. The measured radii of gyration do not change significantly with salt concentration. Small-angle neutron scattering, together with calculations from a Monte Carlo model, allows to determine the superhelix diameter. Measured and simulated scattering curves agreed almost quantitatively. Experimentally we find that the diameter decreases from 16.0±0.9 nm at 10 mM to 9.0±0.7 nm at 100 mM NaCl. The superhelix diameter from the simulated conformations decreased from 18.0±1.5 nm at 10 mM to 9.4±1.5 nm at 100 mM NaCl. At higher salt concentrations up to 1.5 M NaCl, the diameter stays constant at 9 nm.This revised version was published online in October 2005 with corrections to the Cover Date.     

Purification and partial characterization of the DNA-dependent RNA polymerase from Rickettsia prowazekii

The DNA-dependent RNA polymerase was purified from Rickettsia prowazekii, an obligate intracellular bacterial parasite. Because of limitation of available rickettsiae, the classical methods for isolation of the enzyme from other procaryotes were modified to purify RNA polymerase from small quantities of cells (25 mg of protein). The subunit composition of the rickettsial RNA polymerase was typical of a eubacterial RNA polymerase. R. prowazekii had beta' (148,000 daltons), beta (142,000 daltons), sigma (85,000 daltons), and alpha (34,500 daltons) subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The appropriate subunits of the rickettsial RNA polymerase bound to polyclonal antisera against Escherichia coli core polymerase and E. coli sigma 70 subunit in Western blots (immunoblots). The enzyme activity was dependent on all four ribonucleoside triphosphates, Mg2+, and a DNA template. Optimal activity occurred in the presence of 10 mM MgCl2 and 50 mM NaCl. Interestingly, in striking contrast to E. coli, approximately 74% of the rickettsial RNA polymerase activity was associated with the rickettsial cell membrane at a low salt concentration (50 mM NaCl) and dissociated from the membrane at a high salt concentration (600 mM NaCl).     

Separation of plasmid DNA topoisomers by multimodal chromatography

The ability to analyze the distribution of topoisomers in a plasmid DNA sample is important when evaluating the quality of preparations intended for gene therapy and DNA vaccination or when performing biochemical studies on the action of topoisomerases and gyrases. Here, we describe the separation of supercoiled (sc) and open circular (oc) topoisomers by multimodal chromatography. A medium modified with the ligand N-benzyl-N-methyl ethanolamine and an elution scheme with increasing NaCl concentration are used to accomplish the baseline separation of sc and oc plasmid. The utility of the method is demonstrated by quantitating topoisomers in a purified plasmid sample.     

The effect of low ionic strength on the radiation chemistry and physical properties of calf thymus DNA

Studies of ultraviolet and circular dichroism spectra of aqueous solutions of calf thymus (CT) DNA confirm the tendency of DNA to change conformation at low ionic strength. The qualitative shape and transition width of 260 nm melting curves below 1 mM NaCl differed significantly from those previously published for DNA solutions containing 1 mM NaCl and above. Neutral aqueous solutions of CT DNA at low ionic strengths (0.1 mM-10 mM NaCl) were irradiated with low doses of gamma-rays. The melting temperature, Tm, of irradiated DNA samples increased below 1 mM NaCl suggesting interstrand crosslinking of the denatured DNA or formation of regions of more thermally stable DNA conformation. The magnitudes of these radiation responses were found to be a function of the time elapsed between salt concentration changes and irradiation as well as time after irradiation. These results are consistent with the hypothesis that the purine and pyrimidine base chromophores in double stranded DNA are sheltered from radical attack by the sugar phosphate backbone. Low dose radiation studies (0.8-8.0 Gy) of CT DNA in 1 mM NaCl and below showed a split dose and dose rate dependence for the sample melting curves.     

Studies on the action and chemical nature of the nuclear factor promoting degradation of chromatin DNA

Chromatin DNA of liver and kidney, obtained by the method of Dingman & Sporn, is inaccessible in 0.14 M NaCl to pancreatic DNase and cytoplasmic DNase. Under the combined action of DNase and nuclear extract (NE) (extraction with 0.14 M NaCl) on chromatin, the DNA of the latter is intensively degraded. The action of NE is tissue-specific—liver NE has almost no effect on kidney chromatin DNA degradation. The removal of protein or RNA from NE deprives it of its ability to accelerate chromatin DNA degradation by DNases. It is assumed that the active part of NE is a complex of a protein and RNA. Here, tissue specificity is determined by both components of this complex. The biological role of the nuclear factor promoting chromatin DNA degradation is not known at present.     

Two-dimensional DNA typing

DNA typing based on gel electrophoretic separation of DNA fragments, followed by hybridization analysis, has become an important analytical tool in areas ranging from forensic science to population biology. This approach can be extended by combining size separation with sequence-specific separation in denaturing gradient gels; this creates a high resolution two-dimensional pattern. The high information content of this system means that very closely related individuals (even monozygotic twins) can be distinguished and that the genetic events associated with development or cancer, for instance, can be followed. Ultimately, 2-D DNA typing could lead to computerized matching of a single individual's genome to a database of genetic markers.     

Protein . nucleic-acid reaction kinetics. Theoretical analysis of the binding reaction between DNA and RNA polymerase.

This paper presents methods developed in order to analyze experimental results concerning the binding of Escherichia coli DNA-dependent RNA polymerase to DNA at high and at low DNA concentrations, using the filter retention assay. The basis hypotheses, under which the mathematical expressions for describing the kinetics of binding are derived, are as follows. (a) At low DNA concentration: equivalence and independence of the specific binding sites; first-order dependence of the binding reaction on both DNA and protein concentration. (b) At high DNA concentration: equivalence and independence of the non-specific binding sites; no direct transfer or one-dimensional sliding of the protein along the DNA. Comparison between theoretical predictions and experimental results at high DNA concentration will allow one to determine the relative value of the rates of binding of RNA polymerase to different promoters (between 1 and 2 in T5 DNA). Binding experiments performed at low DNA concentration are reported in this paper: these results and the analysis which is reported allow one to determine the value of the rate constant of formation of non-filterable complexes for the system fd DNA (replicative form) . RNA-polymerase (kappa a = 3.3 X 10(8) M-1 s-1 in 0.1 M NaCl, 0.01 M MgCl2).     

Contribution of the resonance Raman spectroscopy to the identification of Z DNA

Poly(dG-dC).poly(dG-dC) at low salt concentration (0.1 M NaCl) and at high salt concentration (4.5 M NaCl) has been studied by Raman resonance spectroscopy using two excitation wavelengths: 257 nm and 295 nm. As resonance enhances the intensity of the lines in a proportion corresponding to the square of the molar absorption coefficient, the intensities of the lines with 295 nm wavelength excitation are enhanced about sevenfold during the B to Z transition. With 257 nm excitation wavelength the 1580 cm-1 line of guanosine is greatly enhanced in the Z form whereas with 295 nm excitation several lines are sensitive to the modifications of the conformation: the guanine band around 650 cm-1 and at 1193 cm-1 and the bands of the cytosines at 780 cm-1, 1242 cm-1 and 1268 cm-1. By comparison with the U.V. resonance Raman spectra of DNA, we conclude that resonance Raman spectroscopy allows one to characterize the B to Z transition from one line with 257 nm excitation wavelength and from three lines with 295 nm excitation. The conjoined study of these four lines should permit to observe a few base pairs being in Z form in a DNA.