Transient electric dichroism studies of the structure of the DNA complex with intercalated drugs.
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Acridines↗
Biomedical subjects
D M Crothers
Publications and source records attributed to D M Crothers.
31P NMR studies of the solution structure and dynamics of nucleosomes and DNA.
31P NMR studies of 140 base pair DNA fragments in nucleosomes and free in solution show no detectable change in the internucleotide 31P chemical shift or linewidth when DNA is packaged into nucleosomes. Measurements of 31P spin-lattice relaxation times T1 and 31P-[H] nuclear Overhauser enhancements revealed internal motion with a correlation time of about 4 x 10(-10) sec in double helical DNA, both free in solution and bound to nucleosomal core proteins. This result implies greater dynamic mobility in double helical DNA than has previously been supposed.
Chromatin↗
Transient electric dichroism studies of nucleosomal particles.
We report transient electric dichroism experiments on nucleosomal core particles containing 140 and 175 base pairs of DNA, and on spacerless dinucleosomes. The results indicate that all particles posses a permanent dipole moment. The orientation time of 140 base pair nucleosomes implies an estimated maximum dimension of a = 130 A (a must be at least 111 A), consistent with the disk model. The maximum dimension of the spacerless dinucleosome is estimated to be about 290 A (at least 180 A), ruling out a structure in which two disks are stacked directly on top of each other. The reduced dichroism amplitude indicate that the DNA superhelix axis in nucleosomes aligns perpendicular to the electric field, as expected for a dipole moment directed along a C2 symmetry axis across the disk diameter. Nucleosomes containing 175 base pairs of DNA show a substantially larger dichroism amplitude that do 140 base pair nucleosomes. In the context of the disk model, this result is shown to be consistent with 100 base pairs of DNA per superhelical turn, but not with 80 base pairs per turn.
Animals↗
Physical studies of nucleosome assemble.
Biochemical, spectroscopic, and hydrodynamic studies were performed on the reconstituted complex of 140 base pair DNA and the arginine-rich histone tetramer (H3/H4)2. The histones bind to DNA in a 1:1 molar ration to form a stable particle which orients in an electric field with a rotational correlation time of 6.3 mus and a limitign reduced dichroism of --0.74. The complex was modeled hydrodynamically as a cylinder of dimensions 450 X 80 X 80 A containing approximately 1.5 superhelical turns. Addition of the lysinerich histones to this complex cause a condensation of the structure and results in physical properties nearly identical with those of a native nucleosomal particle.
Animals↗
Transient electric dichroism of rod-like DNA molecules.
We report transient electric dichroism studies on monodisperse rod-like DNA molecules. By using restriction fragments and DNAs of known length, it is shown that the orientation time is accurately predicted by the theoretically calculated rotational diffusion coefficient. The field dependence of the steady-state dichroism values is not consistent with the induced electric dipole orientation mechanism, and the time dependence is not consistent with the presence of a permanent dipole moment. In order to explain the dependence of the dichroism on the electric field, the ionic strength of the medium, and the length of the macromolecule, we propose a new model in which anisotropic ion flow produces an asymmetric ion atmosphere around the polyelectrolyte, resulting in an orienting torque. From the limiting dichroism at high field, we estimate that the DNA bases are inclined at an angle of 73 degrees or less relative to the helix axis, in good agreement with the revised model of B-form DNA suggested by Levitt, in which the base pairs have a propeller-like twist. Our results establish transient electric dichroism measurements as a technique well suited for study of alterations in the length and base pair inclination of rod-like DNA molecules.
Anions↗
On the physical basis for ambiguity in genetic coding interactions.
We report the relative stabilities, in the form of complex lifetimes, of complexes between the tRNAs complementary, or nearly so, in their anticodons. The results show striking parallels with the genetic coding rules, including the wobble interaction and the role of modified nucleotides S2U and V (a 5-oxyacetic acid derivative of U). One important difference between the genetic code and the pairing rules in the tRNA-tRNA interaction is the stability in the latter of the short wobble pairs, which the wobble hypothesis excludes. We stress the potential of U for translational errors, and suggest a simple stereochemical basis for ribosome-mediated discrimination against short wobble pairs. Surprisingly, the stability of anticodon-anticodon complexes does not vary systematically on base sequence. Because of the close similarity to the genetic coding rules, it is tempting to speculate that the interaction between two RNA loops may have been part of the physical basis for the evolutionary origin of the genetic code, and that this mechanism may still be utilized by folding the mRNA on the ribosome into a loop similar to the anticodon loop.
Base Sequence↗
Does irehdiamine kink DNA?
We report equilibrium, relaxation kinetic, and transient electric dichroism studies on the complex of the diamino steroid irehdiamine A with DNA. The results are consistent with a beta-kinked structure for the complex at saturation, with a kink in the DNA structure induced by a bound steroid every second base pair. The results that favor this hypothesis include an apparent length decrease of rod-like bacterial DNA molecules when only a small amount of drug is bound, followed by an apparent length increase at saturation. The limiting dichroism amplitude implies a substantial increase in the tilt of the bases relative to the orientation axis; at saturation the base UV transition moments are tilted about 31 degrees from the plane perpendicular to the orientation axis. Because of the direction of polarization of the 260-nm transition moments, the results indicate that the tilt of the bases must be predominantly in the short rather than the long axis of the base pair. The large hyperchroism of the complex is consistent with loss of base stacking, as required by a kinked structure. The kinetic results imply a bimolecular reaction mechanism, with a temperature-dependent association rate constant of roughly 10(8) M(-1) sec(-1), and a dissociation rate constant of about 5 x 10(3) sec(-1), nearly independent of temperature. The association activation energy and apparent reaction enthalpy vary from 12 to 22 kcal mol(-1); heat is absorbed on complex formation as expected for loss of base-stacking interactions. An anomalous result of the experiments is the larger apparent length increase (13%) exhibited by two eukaryotic DNAs, compared to 6% for three prokaryotic DNAs. Differences were also observed in the kinetic properties of the complexes.
Binding Sites↗
Preparative separation of the complementary strands of DNA restriction fragments by alkaline RPC-5 chromatography.
High pressure reversed phase chromatography (RPC-5) at pH 12 was used for preparative separation of the complementary strands of the smaller DNA fragments which are generated by the Hae III restriction endonuclease digestion of Col El DNA. A single high pressure RPC-5 chromatographic step at neutral pH served to purify duplex fragments 70, 172, 250 and 440 base pairs long; each of these yielded two elution peaks upon chromatography under alkaline denaturing conditions.
Bacteriocin Plasmids↗
Simplified methods for large scale enzymatic synthesis of oligoribonucleotides.
We report simplified methods for large scale enzymatic synthesis of oligoribonucleotides using polynucleotide phosphorylase. The main features of the method are use of RPC-5 chromatography, including chromatography at two pH values to deal with the problem of primer phosphorolysis, rapid dialysis for large scale desalting, simplified methods for enzyme removal, and high resolution 1H and 31P NMR for product identification and demonstration of purity. The capacity of the method is adequate to allow beginning with grams of material in the first polymerization step, so that product yields of several milligrams, sufficient for many physical studies, are possible after as many as three separate polymerization reactions.
Base Sequence↗
Electric dichroism studies of the size and shape of nucleosomal particles.
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Animals↗
Influence of the A15 mutation on the conformational energy balance in Escherichia coli tRNA Tyr.
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Escherichia coli↗
Does 5S RNA function by a switch between two secondary structures?
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Models, Structural↗
Pathway-dependent refolding of E. coli 5S RNA.
The refolding of 5S RNA into its two conformational states has been examined as a function of solvent composition and annealing conditions. The results show that the product distribution depends on the folding pathway. Quick cooling from high temperature produces roughly equal amounts of the two forms, even in the presence of 1 mm Mg++. However annealing by slow cooling to intermediate temperatures (50 degrees--60 degrees C) in Mg++-containing buffers, followed by quick cooling, allows formation of a structure which guides the refolding path to the "native" conformation. The stability of this structural nucleus for the "native" conformation depends strongly on Mg++ concentration. We conclude that the A ("native") conformation differs from the B conformation not in rate of refolding, but rather in having a lower enthalpy and a also a smaller rate of unfolding for the critical structural nucleus. The order of folding during biosynthesis may be crucial for forming the "native" conformation.
Escherichia coli↗
Isolation and characterization of a spacerless dinucleosome from H1-deleted chromatin.
Calf thymus chromatin, depleted in histone H1, was digested with micrococcal nuclease and fractionated by column chromatography. 140 base pair nucleosome core particles were isolated along with an unusual particle containing 2 histone octamers and 240 base pairs of DNA. Evidence is presented that the spacer DNA region is absent from these modified dinucleosomes, which appear as stable products of the digestion process. The physical properties of both particles are presented along with brief speculation on their possible origin and function.
Animals↗
An ethidium-induced double helix of poly(dA)-poly(rU).
Equilibrium dialysis, relaxation kinetic, melting, and continuous variation mixing experiments on complexes of poly(dA) and poly(rU) demonstrate that ethidium induces conversion of a 1:1 mixture of these homopolymers (at one molar salt and 19 degrees C) from a three stranded to a two stranded helix. This is the first demonstration of a double helix of poly(dA)-poly(rU) in solution.
Binding Sites↗
Kinetic studies of drug-dinucleotide complexes.
Three classes of kinetic behavior are observed in the complexes of actinomycin or ethidium with deoxydinucleotides. First, the initial dinucleotide binding to form a 1:1 complex is a rapid bimolecular process, whose rate could be measured for combination of actinomycin with d(pTpG) d(pGpT), d(pGpA), d(pGpG) d(pCpGpG), and d(pCpG) andfor combination of ethidium with d(pGpC). Second, with one exception, all reactions in which a second dinucleotide is added to form a 2:1 dinucleotide-drug complex are limited by a first-order step at high concentration. This class includes the combination of actinomycin with all dinucleotides tested except d(pGpC), and the reaction of ethidium with nucleotides of complementary sequence pyrimidine-purine, such as d(pCpG). The final class is the special case of d(pGpC) interacting to form a 2:1 complex with actinomycin. Third-order kinetics is observed, with no evidence for a first-order, rate-limiting step.
Chemical Phenomena↗
Physical characterization of a ribosomal nucleoprotein complex.
The complex between ribosomal protein L24 and its RNA binding site (that region of the 23S RNA which the protein protects from ribonuclease digestion) has been studied by various physicochemical methods. The RNA is composed of two fragments of about 160 and 140 nucleotides which interact with each other to form the L24 binding site. Circular dichroism spectroscopy suggests that the two interacting fragments have a unique region of secondary structure which is not present in either of the two components alone; hence there are important structural interactions between regions of the RNA which are separated in the primary sequence. Addition of the L24 protein to the RNA site promotes a structural change associated with base unstacking, but with little or no change in the hydrogen-bonded base pairing. Heat activation is not required for complex formation. Thermal denaturation studies reveal a broad featureless transition and the amount of hypochromic change indicates that the RNA site contains less secondary structure than other RNAs such as tRNA and total rRNA. Temperature-jump relaxation measurements on the mechanism of unfolding of the RNA show a concerted melting of the entire secondary and tertiary structure, which is altered upon addition of the protein. A structrual basis for this RNA-protein complex is discussed.
Circular Dichroism↗
Phase partition studies of actinomycin-nucleotide complexes.
A method is reported for measuring the stoichiometry of complex formation between actinomycin and a series of deoxynucleotides. The amount of bound actinomycin is measured by distribution of the drug between two liquid phases, a buffer phase containing deoxynucleotide and an organic phase in which the nucleotide is insoluble. Using simple statistical mechanical analysis, the equilibrium equations for several models of actinomycin-deoxynucleotide complexes have been derived: actinomycin with one binding site, with two equivalent independent binding sites, and with two sites which must be occupied together. The binding of actinomycin C3 with dpG, dpApG, dpA, and dpGpC has been examined compared with these models. It is found that binding to dpG and dpApG involves two independent binding sites of nearly equal affinity for nucleotides, whereas binding of dpGpC to the two binding sites on actinomycin is a cooperative process. Binding of dpA tp actinomycin is partially cooperative and weaker than binding of dpG. The dimerization constant of actinomycin was also determined by the phase separation technique, and found in agreement with other values, including the results of kinetic measurements reported here.
Binding Sites↗