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G L Eichhorn

Publications and source records attributed to G L Eichhorn.

At least 19 recordsLinked to original sources
PubMed1999

Curvature of dinucleotide poised for formation of trinucleotide in transcription with Escherichia coli RNA polymerase.

A frequently used schematic model of transcriptional elongation shows an RNA polymerase molecule moving along a linear DNA. This model is of course highly idealized and not compatible with promoter sequences [Gralla, J. D. (1991) Cell 66, 415-418; Schleif, R. (1992) Annu. Rev. Biochem. 61, 199-223] and regulatory proteins [Koleske, A. J., and Young, R. A. (1995) Trends Biochem. Sci. 20, 113-116; Dunaway, M., and Dröge, P. (1989) Nature 341, 657-659; Müller, H. P., Sogo, J. M., and Schaffner, W. (1989) Cell 58, 767-777] located some distance away from the point of transcription initiation [Karsten, R., von Hippel, P. H., and Langowski, J. (1995) Trends Biochem. Sci. 20, 500-506]. These circumstances lead to the expectation of curvature along the DNA strand and require looping between sometimes distant points. We have now shown curvature in a dinucleotide formed at the very onset of transcription when it is poised for reaction with a mononucleotide to form a trinucleotide. The curvature became evident from the demonstration that a metal ion bound with a mononucleotide in the i+1 (elongation) site is approximately equidistant from bases at the 5' end (i-1 site) and 3' end (i site) of the dinucleotide. Similar results were obtained with three different dinucleotides and four mononucleotides. Curvature of the RNA initiate may reflect curvature of the DNA to which it is bound. These studies show curvature to be a significant feature in the interaction between DNA template and RNA elongate even at the very beginning of transcription.

Adenosine Triphosphate↗
PubMed1997

Specificity of an Escherichia coli RNA polymerase-associated NTPase.

Standard preparations of Escherichia coli RNA polymerase harbor a 70 kDa protein with NTPase (beta-gamma cleavage) activity that is not a recognized polymerase subunit. The NTPase activity of this component, before and after separation from the polymerase, is strongly dependent on the presence of DNA; single-stranded polydeoxynucleotides are more effective than double-stranded. ATP and GTP are cleaved, the latter much less readily. The NTPase as it occurs with the polymerase displays cleavage preference for NTPs that are not complementary to the DNA, a fact that has led to proposals for involvement of the NTPase in transcriptional error prevention [Volloch, V. Z., Rits, L. & Tumerman, L. (1979) Nucleic Acids Res. 6, 1535-1546; Libby, R. T., Nelson, J. L., Calvo, J. M., & Gallant, J. A. (1989) EMBO J. 8, 3253-3158]. We find, however, that the lesser cleavage in the presence of complementary DNA results from competition for the NTP between the processes of incorporation by the polymerase and of cleavage by the NTPase, operating on the same substrate pool. The greater cleavage with noncomplementary DNA occurs because of the lack of incorporation by the polymerase, which then does not compete with the NTPase for the substrate pool. Thus, these findings indicate that the cleavage preference of the NTPase for noncomplementary NTPs is not part of a mechanism for error prevention during transcription.

Acid Anhydride Hydrolases↗
PubMed1995

A simple probe for DNA accessibility in chromatin.

When DNA is treated with Cu(II) and then heated, the melting temperature (Tm) of the DNA is dramatically decreased (8). The Cu(II) binds to the DNA in such a way as to destabilize the double helix and help to break the hydrogen bonds between the bases. When soluble chromatin is similarly treated with Cu(II) and heated, the Tm is unaffected. Apparently the Cu(II) cannot penetrate the chromatin structure and thus cannot initiate the DNA destabilization process. However, when H-1 histone is removed from the chromatin by affinity chromatography, subsequent treatment with Cu(II) does lead to a lowered Tm when the chromatin is heated. This Tm lowering is also achieved by two less drastic techniques that do not remove histone H-1, but decrease the affinity of the H-1 to the DNA: (1) a mild acetylation procedure that specifically modifies either 2 or 4 epsilon-amino groups of lysines on the H-1 histone, and (2) reaction with phosphate-binding divalent metal ions, e.g., Mg(II), Mn(II), or Co(II). Apparently, removal of H-1 or decreased affinity of H-1 for DNA increases the accessibility of the DNA to the Cu(II). This phenomenon suggests a very simple qualitative probe for the degree of structural change in chromatin produced by a change in the stability of the DNA-H-1 interaction.

Acetylation↗
PubMed1994

A structural model for fidelity in transcription.

Distances between the metal ions bound to the product terminus i site and the substrate i + 1 site of Escherichia coli RNA polymerase range from 5.0 to 5.6 A when the substrate is complementary to a template base and from 6.5 to 7.0 A for a noncomplementary relationship. The metal bound to the substrate at the i + 1 site exhibits a constant distance to the three phosphates on the substrate regardless of complementarity, but the distance to base and ribose protons changes. The differences in these geometric parameters are explained by the ability of the enzyme to assume two conformations, one to place correct nucleotide substrates in optimal position for bond formation and the other to prevent incorrect nucleotides from assuming such a position. In this scheme a metal-triphosphate complex can move toward or away from the terminal 3' OH group of the growing RNA chain, to assure fidelity of transcription.

Base Composition↗
PubMed1991

Selectivity of Escherichia coli RNA polymerase for template conformation.

Escherichia coli RNA polymerase (RNAP) exhibits a strong selectivity for the secondary structure of its template DNA, as shown by the influence both of the DNA conformation on the transcription cycle and of the enzyme on the DNA conformation itself. Binding, chain initiation and elongation characteristics of RNAP, and DNA conformational characteristics were examined by use of the alternating copolymer poly(dGdm5C).poly(dGdm5C) as template. Transcription is impeded when the DNA is in the Z conformation as compared with the B; the initial conformation is determined by the concentration of the conformational effectors of Mg2+ and [Co(NH3)6]3+. RNAP binds to both Z and B conformers; the total binding is moderately greater when the template is in the B conformation than when it is strongly stabilized in the Z, by [Co(NH3)6]3+ concentrations much higher than those required for B-Z transition. However, the Z conformer is much more easily displaced competitively from the bulk of its complexes with RNAP than is the B, indicating a specific binding preference for the B conformer. When the template is in the B conformation, or is moderately stabilized in the Z by Mg2+ concentrations such that the polynucleotide is just fully converted from B to Z, elongation is predicted well by chain initiation, indicating that on the Z conformer RNAP is effectively inhibited at the chain initiation or at an earlier stage. The average chain growth rates for polymeric product synthesized on B and on moderately stabilized Z are similar, even though overall RNA synthesis is considerably lowered on the Z form, again indicating that the limiting events precede elongation. When the Z conformer is strongly stabilized, chain initiation and elongation are further inhibited. Elongation is still roughly correlated with chain initiation, but some additional inhibition of elongation takes place independently. Circular dichroism analysis shows that RNAP-DNA binding affects the B-Z conformational equilibrium, leading to reformation of the B conformer from Z and interference with conversion of B to Z, under conditions that would otherwise favor the Z conformer. Thus, there is an RNAP concentration dependent shift of the B-Z transition to higher concentrations of Z-inducing cation, and there is an RNAP concentration dependent decrease in the rate of B to Z conversion. These effects were observed for poly(dGdm5C).poly(dGdm5C), with Z stabilized by [Co(NH3)6]3+ or Mg2+. (They were observed as well for the unmethylated copolymer poly(dGdC).poly(dGdC), with Z stabilized by [Co(NH3)6]3+.) Perturbation of the Z conformer was detectable by circular dichroism at an RNAP:polynucleotide ratio down to a practical limit of approximately 1 RNAP:500 bp.(ABSTRACT TRUNCATED AT 400 WORDS)

Circular Dichroism↗
PubMed1990

Structural studies on the active site of Escherichia coli RNA polymerase. 1. Interaction of metals on the i and i + 1 sites.

The two substrates between which an internucleotide bond is formed in RNA synthesis occupy two subsites, i and i + 1, on the active site of Escherichia coli RNA polymerase, and each subsite is associated with a metal ion. These ions are therefore useful as probes of substrate interaction during RNA synthesis. We have studied interactions between the metals by EPR spectroscopy. The Zn(II) in the i site and the Mg(II) in the i + 1 site were substituted separately or jointly by Mn(II). The proximity of the metals was established by EPR monitoring of the titration at 5.5 K of the enzyme containing Mn(II) in i with Mn(II) going into the i + 1 site, and the 1:1 ratio of the metals in the two sites was confirmed in this way. The distance between the two metals was determined by EPR titration at room temperature of both the enzyme containing Zn(II) in i and Mn(II) in i with Mn(II) going into the i + 1 site, making use of the fact that EPR spectra are affected by dipolar interactions between the metals. The distances calculated in the presence of enzyme alone, in the presence of enzyme and two ATP substrates, and when poly(dAdT).poly(dAdT) was added to the latter system ranged from 5.2 to 6.7 A.

Adenosine Triphosphate↗
PubMed1990

Structural studies on the active site of Escherichia coli RNA polymerase. 2. Geometrical relationship of the interacting substrates.

Since a major function of RNA polymerase must be to bring together substrates in the optimal configuration for internucleotide bond formation, studies have been undertaken to understand the geometrical relationship of the two substrates. A model has been constructed for the geometry of interaction of two ATP molecules poised on the active site of the Escherichia coli enzyme for the formation of the first bond in RNA synthesis. The model is based primarily on the distance, measured by EPR, between the two metals in the i and i + 1 subsites, as well as distances, measured by NMR, from each metal to points on the substrate in the same subsite, in the presence of a poly(dAdT).poly(dAdT) template. Both the Zn(II) in the i site and the Mg(II) in i + 1 are displaced by Mn(II). The nucleotide bases are not parallel to each other, in line with the reaction of the ATP molecules with DNA within the transcription bubble. The metal in the i site appears too far removed from substrate to participate in catalysis, but the metal in i + 1 is in position to bind to the beta- and gamma-phosphate groups and probably is involved in cleavage of the triphosphate, as has been previously suggested.

Adenosine Triphosphate↗
PubMed1989

Polynucleotide cross-linking by aluminum.

The observations that there was an increased concentration of Al in the brains of Alzheimer's, Guam-Parkinson, and amyotrophic lateral sclerosis disease patients and that there was an apparent localization of the Al in chromatin led to a study of the interaction of Al(III) with DNA. We have previously shown that Al cross-links calf thymus DNA at low pH (S. J. Karlik, G. L. Eichhorn, P. N. Lewis, and D. R. Crapper, Biochemistry 19, 5991 [1980]). Extended studies indicate that cross-linking occurs in DNAs of all base ratios, including polydAdT and polydGdC. Since Al cross-links prevent renaturation in polydAdT, the decrease in the amount of polymer renatured in the presence of Al becomes a quantitative appraisal of the extent of cross-linking. Saturation of cross-linking occurs at a 0.4 ratio of Al to nucleotide phosphate, indicating that potentially 80% of the base pairs are Al bound. Cross-links are broken at elevated pH and by EDTA.

Aluminum↗
PubMed1989

Reversible toroidal compaction of DNA by aluminum.

Electron microscopy has been used to characterize the products of the reactions of aluminum with DNA under three different conditions, one of which, pH 5 and Al (III)/DNA(P) ratio of 0.4, has been previously shown to produce reversible interstrand crosslinking in double-stranded DNA molecules. Under this condition, aluminum produced macromolecular aggregates of DNA upon heating, with a distinctive ultrastructure reversible to double-stranded DNA after removal of the aluminum. These structures were toroidal in configuration and exhibited mean widths of 4.9 +/- 1.8 nm and were 18.6 +/- .4 nm in diameter with a toroidal internal diameter of 7.4 +/- 4.7 nm. Previous results have shown that these structures contain Al(III) - crosslinked DNA, the present data suggests that this intermolecular crosslinking is associated with the production of compacted structures.

Aluminum↗
PubMed1987

DNA methylation in aging of mice.

Methylation of cytidine residues of DNA appears to be involved in the control of gene expression; therefore, hypomethylation of the DNA can be considered to be an active rather than a passive process. Previous studies of mammalian DNA methylation during aging have produced an assortment of results. In this study, we have examined the change in DNA base composition, including the change in 5-methyldeoxycytidine (m5dC) contents with age of mice. Livers pooled from 6 mice from each of six age groups between 6 and 31 months have been subjected to a sensitive analytical technique (HPLC). The DNA composition of different age groups is very consistent in most aspects. The ratio of (dA + dT)/(dG + dC + m5dC) as well as the sum of dC and m5dC remain constant throughout the animal's lifespan. However, a consistent gradual decline in m5dC content is noted as the age increases to 24 months. Thus, the 6-month-old animal pool exhibits the largest amount of m5dC (1.67 +/- 0.2%), which is reduced consistently as the animal's age reaches 24 months. This decrease in m5dC is accompanied by an increase in dC. No further decrease in m5dC occurs after 24 months; in fact, the data could indicate an increase after that age. No dTs are apparently produced by deamination of m5dC.

Aging↗
PubMed1987

The effect of cellular age on zinc levels in untreated and zinc-treated human diploid fibroblasts.

Cellular aging is accompanied by increased cellular permeability to zinc(II). The intrinsic zinc content of human diploid fibroblast cells increases with cell age, so that it quadruples from early to late passage, on a Zn(II) per cell or per cell volume basis, but it remains constant on a Zn(II) per protein basis. When the cells are challenged with toxic concentrations (0.2 mM) of Zn(II), both the rate of zinc incorporation into the cells and the amount of zinc incorporated at equilibrium increases considerably with age (unless measured as zinc per protein). In terms of growth inhibition, Zn(II) is more toxic to the cell than Cu(II), Mn(II), or Mg(II).

Cations, Divalent↗
PubMed1986

31P NMR spectroscopy of isolated perfused lungs.

31P NMR spectra of isolated blood-perfused pig lungs were obtained by degassing the lungs in vivo to remove field inhomogeneities caused by air-tissue interfaces. The spectra show the presence of ATP, phosphodiester, inorganic phosphate, and phosphomonoester, but no phosphocreatine. All the metabolites remained stable for more than 4 h when the lungs were perfused with oxygenated blood. Blood gas tensions, glucose concentration, pH, and temperature were controlled throughout the experiment. During anoxia or ischemia, ATP and intracellular pH declined and Pi increased but returned to control levels during subsequent normoxia or reperfusion. These results demonstrate the applicability of NMR spectroscopy to isolated perfused lungs, enabling studies of metabolic processes in normal and pathologic lungs, as well as establishment of optimal conditions for lung preservation for transplantation.

Animals↗
PubMed1986

Factors controlling the Pb(II) promoted dephosphorylation of nucleotides.

The conditions under which Pb(II) promotes dephosphorylation of nucleotides have been studied with the Pb(II) complexes of several isomers of AMP, dAMP, GMP, and dGMP. A number of factors which together control the dephosphorylation reaction have been identified. These include the tendency of Pb(II)-induced nucleotide base stacking, as evidenced by large enhancement in ultraviolet circular dichroism, to occur in the complexes and limit the reaction; hydroxylation of the metal, either with weakening of the lead-nucleotide binding, or eventually with displacement of the nucleotide; and the solubility of the complexes, which limits the reaction, but is increased by raising the temperature and by hydroxylation of the complexes. The pH range in which both base stacking and metal hydrolysis are minimized can define a "reaction window" for the complexes.

Chemical Phenomena↗
PubMed1984

Effect of template conversion from the B to the Z conformation on RNA polymerase activity.

Transition from the right-handed B to the left-handed Z conformation of DNA was studied by circular dichroism in parallel with the ability of the DNA to support RNA synthesis with Escherichia coli RNA polymerase. Since the B to Z transition is generally induced by a chemical agent, a definitive demonstration that a change in activity is due to the conformational change, and not to the agent itself or to other factors, requires the clear-cut correlation of template activity and conformation under a variety of conditions that result in conformational change. Such correlation was achieved by following the [Co(NH3)6]3+-induced transition of poly(dG-dC) X poly(dG-dC) and poly(dG-dm5C) X poly(dG-dm5C) and the Mg2+-induced transition of poly(dG-dm5C) X poly(dG-dm5C). In addition, conditions were chosen to minimize possible aggregation. In each of these three systems, the B to Z conformational transition was accompanied by a substantial decrease in transcription activity. While the conversion from B to Z of poly(dG-dm5C) X poly(dG-dm5C) is induced by a 25-fold lower concentration of [Co(NH3)6]3+ than that required for the conversion of unmethylated polymer, in both cases the RNA polymerase activity is decreased at the same cation concentration as that producing the conformational transition. Neither [Co(NH3)6]3+ nor Mg2+ inhibits RNA synthesis with control templates that are not converted to Z under the same conditions, such as poly(dA-dT) X poly(dA-dT) or calf thymus DNA with [Co(NH3)6]3+ or poly(dG-dC) X poly(dG-dC) with Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗