Temperature dependence of the rate constants of the Escherichia coli RNA polymerase-lambda PR promoter interaction. Assignment of the kinetic steps corresponding to protein conformational change and DNA opening

The kinetics of formation and of dissociation of open complexes (RPo) between Escherichia coli RNA polymerase (R) and the lambda PR promoter (P) have been studied as a function of temperature in the physiological range using the nitrocellulose filter binding assay. The kinetic data provide further evidence for the mechanism R + P in equilibrium I1 in equilibrium I2 in equilibrium RPo, where I1 and I2 are kinetically distinguishable intermediate complexes at this promoter which do not accumulate under the reaction conditions investigated. The overall second-order association rate constant (ka) increases dramatically with increasing temperature, yielding a temperature-dependent activation energy in the range 20 kcal (near 37 degrees C) to 40 kcal (near 13 degrees C) (1 kcal = 4.184 kJ). Both isomerization steps (I1----I2 and I2----RPo) appear to be highly temperature dependent. Except at low temperatures (less than 13 degrees C) the step I1----I2, which we attribute to a conformational change in the polymerase with a large negative delta Cp degrees value, is rate-limiting at the reactant concentrations investigated and hence makes the dominant contribution to the apparent activation energy of the pseudo first-order association reaction. The subsequent step I2----RPo, which we attribute to DNA melting, has a higher activation energy (in excess of 100 kcal) but only becomes rate-limiting at low temperature (less than 13 degrees C). The initial binding step R + P in equilibrium I1 appears to be in equilibrium on the time-scale of the isomerization reactions under all conditions investigated; the equilibrium constant for this step is not a strong function of temperature and is approximately 10(7) M-1 under the standard ionic conditions of the assay (40 mM-Tris . HCl (pH 8.0), 10 mM-MgCl2, 0.12 M-KC1). The activation energy of the dissociation reaction becomes increasingly negative at low temperatures, ranging from approximately -9 kcal near 37 degrees C to -30 kcal near 13 degrees C. Thermodynamic (van't Hoff) enthalpies delta H degrees of open complex formation consequently are large and temperature-dependent, increasing from approximately 29 to 70 kcal as the temperature is reduced from 37 to 13 degrees C. The corresponding delta Cp degrees value is approximately -2.4 kcal/deg. We propose that this large negative delta Cp degrees value arises primarily from the burial of hydrophobic surface in the conformational change (I1 in equilibrium I2) in RNA polymerase in the key second step of the mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)     

Processing enzyme ribonuclease E specifically cleaves RNA I. An inhibitor of primer formation in plasmid DNA synthesis

When the RNA processing enzyme RNAase E is inactivated in an Escherichia coli strain carrying derivatives of the colicin E1 plasmid, a small RNA, about 100 nucleotides long, accumulates. Structural analysis of this RNA showed that it is RNA I, the RNA that inhibits plasmid DNA synthesis. RNA I is a specific substrate for RNAase E and the cleavage takes place between the fifth and sixth nucleotides from the 5' end of the molecule. This is only the second natural RNA substrate that has been found, so far, for the RNA processing enzyme ribonuclease E, the other being a precursor for 5 S ribosomal RNA. It is remarkable that nine nucleotides around the cleavage sites are identical in both substrates: (Formula: see text). Therefore, we suggest that at least part of the interaction between RNAase E and its substrate is controlled by these nine nucleotides.     

Thermodynamics of the reaction of ferric myoglobin from Aplysia limacina with azide and fluoride. Dependence of enthalpy changes on pH

The effect of pH on the enthalpy changes for binding of azide and fluoride to ferric myoglobin from Aplysia limacina, which lacks the distal histidine, has been investigated. Over the whole pH range explored (3.8 to 9.5), -delta H degrees values for the formation of the hemoprotein-ligand complexes are: (1) much greater than the variations in -delta G degrees; (2) always negative; and (3) show a dependence upon pH characterized by a maximum for azide and a minimum for fluoride binding, centered at pH 4.55 (identical to pHch). This value agrees well with that expected from the linear correlation between pHch and the simple function "Lys+Arg-Glu-Asp-2" proposed by Beetlestone and others. Data reported here greatly extend the pH range for which the linear correlation between the net charge of the macromolecule and pHch has been found to hold, and indicate unequivocally that the pH dependence of -delta H degree for the binding of anionic ligands does not uniquely require the presence of the histidyl residue at the distal position.     

Interdependence of growth, water relations and abscisic acid level in Phaseolus vulgaris during waterlogging

The interdependence between changes in growth and water relations after waterlogging was investigated by recording simultaneously growth, transpiration, water potential, turgor, leaf diffusion resistance and abscisic acid content in Phaseolus vulgaris L. cv. bruine Noord-Hollandse. Growth was inhibited immediately after flooding, whereas transpiration decreased gradually to a low level in about three days. The first two days after flooding a small increase in abscisic acid content in the leaves was observed which was accompanied by an increase in diffusion resistance. The increase in abscisic acid content could result from an inhibited export from the leaves. After the first two days a decrease in water potential and turgor was accompanied by a drastic increase in both abscisic acid content and diffusion resistance. This large increase in abscisic acid content occurred before the turgor had reached its minimum value. The change in diffusion resistance kept showing a lag of about one day with the change in abscisic acid content. The possibility is discussed that besides abscisic acid also its metabolite phaseic acid is involved in stomatal closure. After the formation of adventitious roots on the hypocotyl, abscisic acid level, diffusion resistance, water potential and turgor returned to the control values. Transpiration showed a slow recovery from the sixth day after flooding, whereas growth was inhibited for at least nine days. A remarkable similarity exists between our observations on the responses of bean plants to flooding and the well known responses to drought.     

Pressure chamber measurements using two wheat leaves

Summary The most widely used technique of leaf water potential measurements is with the Scholander pressure chamber. Representative leaf water potential values require many determinations on individual leaves and this can be time consuming in large fields or experiments with multiple treatments. This paper describes a method of obtaining a mean value more rapidly, by using two leaves in the pressure chamber at the same time, but recording the end point of each leaf separately.     

Plastid changes during the conversion of chloroplasts to chromoplasts in ripening tomatoes

Methods were developed for the isolation of plastids from mature green and ripening tomatoes (Lycopersicon esculentum Mill.) and purification by sucrose or Percoll density-gradient centrifugation. Assessment of the purity of preparations involved phase-contrast and electron microscopy, assays for marker enzymes and RNA extraction and analysis. Proteins were extracted from isolated plastids at different ripening stages and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The profiles obtained from chloroplasts and chromoplasts showed many qualitative and quantitative differences. Labelling of proteins with [³⁵S]methionine in vivo showed that there was active protein synthesis throughout ripening, but there was a change in the plastid proteins made as ripening proceeded. The cellular location of synthesis of specific proteins has yet to be established.Abbreviations CS citrate synthase - EDTA ethylenediaminetetraacetic acid,-acetate - GAPDH NADP⁺-glyceraldehyde-3-phosphate dehydrogenase - rRNA ribosomal RNA - SDS sodium dodecyl sulphate - SDS-PAGE SDS-polyacrylamide gel electrophoresis - Tris 2-amino-2(hydroxymethyl)-1,3-propanediol     

Solvent influence on rate and mechanism of oxidation of ethylenediaminetetraacetatocobaltate(II) by periodate

The kinetics of the oxidation of Co^II(EDTA)²⁻ by IO₄⁻ were studied in various ethanol + water mixtures covering the range 7.9 to 58.0 wt% ethanol, at five different temperatures in the range 15–35 °C. The effect of solvent on the rate and mechanism of the reaction was investigated. An inner-sphere mechanism for the reaction was proposed and supported by the calculated activation parameters.     

Stability enhancement and circular dichroism spectral anomaly as an indication of non-covalent interactions in histidine- and tyrosine-containing ternary copper(II)—amino acid systems

Visible absorption and CD spectral and potentiometric studies on the His- and Tyr-containing ternary copper(II) complexes Cu(A)(L-B), where A refers to L-His, D-His, or L-Tyr and B to Lys, Tyr, Trp, Phe, Ala, Val, Arg, Glu, Asn, Gln, Ser, or Thr, were made to study ligand-ligand interactions in the complexes. While the CD spectral magnitudes in the d—d region are additive in the absence of side chain interactions and can be estimated from the magnitudes for the ternary systems involving DL-A or DL-B, deviation from the additivity was observed for Cu(L-His)(L-B) (B = LysH, Tyr, Trp, or Phe) and Cu(L-Tyr)(L-Trp). From the stability constants determined at 25 °C and I = 0.1 M (KNO₃), the equilibrium constants, K, for the following hypothetical equilibria were calculated to be large (0.14–0.60) for formation of Cu(L-/D-His)(L-B)(B = Tyr or Trp) and Cu(D-His)(L-Phe) with Cu(en)(L-Ala) as standard: $Cu(A)(L?Ala)+Cu(en)(L?b)\stackrel{K}{?}Cu(A)(L?B)+Cu(en)(L?Ala)$ The positive values indicate the stabilization due to the stacking between the imidazole ring of His and the aromatic side chain of L-B. Solvent dependence of the CD spectra for Cu(L-His)(L-LysH) and Cu(L-His) L-Trp) further supported the existence of the intramolecular electrostatic and hydrophobic interactions.     

Axial ligation of nitrogenous bases to five-coordinate chloro-meso-tetraphenylporphyrinatochromium(III)

The axial ligations of nitrogenous bases to the five-coordinate chloro-meso-tetraphenylporphyrinatochromium(III) [Cr(III)(TPP)(Cl)] were studied in a non-coordinating solvent, dichloromethane (CH₂Cl₂), by spectrophotometric methods. A correlation exists between log K for the axial ligation:

and pK_a for the N-donor ligand. This correlation suggests that ligand to metal σ bonding contributes to the complex formation, rather than does metal to ligand π back-donation.     

Tetrakis-[1-methylimidazoline-2(3H)-thione]-μ2-[1-methylimidazoline- 2(3H)-thione]-Di-Copper(I) sulphate trihydrate: Preparation, thermal analysis and crystal structure

1-emthylimidazoline-2(3H)-thione (mimtH) reacts with copper(II) sulphate pentahydrate in aqueous acetone to produce the dinuclear complex, Cu₂(mimtH)₅SO₄ · 3H₂O; the formula has been established by a combination of chemical and thermal analysis. The monoclinic crystals, (space group P_c, Z = 2), contain dinuclear cations, sulphate ions and water molecules. The dinuclear cation, Cu₂(mimtH)₅²⁺, consists of two trigonal copper(I) atoms, four terminal, monodentate, S-donating mimtH molecules and one S-bridging (μ₂) mimtH molecule. Some average dimensions are:Cu---S, 2.258 Å and S---Cu---S, 120.0°; the Cu---S---Cu bridging angle is 94.8° and the Cu---Cu separation distance is 3.308 Å.