Kinetics of promoter search by Escherichia coli RNA polymerase. Effects of monovalent and divalent cations and temperature

The rapid mixing/photocross-linking technique developed in our laboratory has been employed in the study of the mechanism of promoter binding by Escherichia coli RNA polymerase (RPase). We have previously reported on the quantitation of the one-dimensional diffusion coefficient (D1) for RPase along the DNA template (Singer, P. T., and Wu, C.-W. (1987) J. Biol. Chem. 262, 14178-14189). In this paper, we describe the effect of salt concentration and temperature on the kinetics of promoter search by RPase using plasmid pAR1319 DNA, which contains the A2 early promoter from bacteriophage T7, as template. Over a range of KCl concentrations from 25 to 200 mM, the apparent bimolecular rate constant (ka) for the association of RPase with the A2 promoter on this DNA template varied approximately 2-fold, achieving a maximal value between 100 and 125 mM KCl. More significantly, the transient distribution of RPase among nonspecific DNA binding sites changed markedly as a function of salt concentration, indicative of gross changes in the average number of base pairs covered by sliding during a nonspecific lifetime. Using the mathematical treatment outlined in our earlier report, the nonspecific dissociation rate constant (koff) was calculated from the binding curves for the nonspecific as well as promoter-containing DNA. The observed variations in ka as a function of monovalent cation concentration ([M+]) were due primarily to changes in koff, as D1 was found to be essentially independent of [M+]. Interestingly, D1 decreased by one-third as the concentration of magnesium was lowered from 10 to 1 mM. In addition, the dependence of koff (and consequently the nonspecific equilibrium association constant, keq) on [M+] agreed qualitatively with the results of deHaseth et al. (deHaseth, P.L., Lohman, T. M., Burgess, R. R., and Record, M. T., Jr. (1977) Biochemistry 17, 1612-1622), though we consistently measure a weaker Keq. The association rate constant was also measured between 4 and 37 degrees C, and was found to vary approximately 2-fold over that range. An activation energy for the bimolecular association of RPase to the A2 promoter was calculated to be 2.2 +/- 0.4 kcal/mol, while the activation energy for one-dimensional diffusion was 4.7 +/- 0.8 kcal/mol.     

Cell damage by cytolysin. Spontaneous recovery and reversible inhibition by divalent cations

Cytolysin-induced membrane damage (which requires low Ca2+) has been studied 1) in E by assay of hemolysis, 2) in Lettre cells by measurement of transmembrane potential, intracellular content of K+ and Na+, leakage of phosphoryl[3H]choline or 51Cr from [3H]choline-labeled or 51CrO4(2-)-labeled cells and leakage of lactate dehydrogenase, and 3) in phospholipid bilayers by measurement of electrical conductivity changes. In Lettre cells, damage is restricted and reversible: little lactate dehydrogenase leaks from cells that leak substantial amounts of Na+, K+, and phosphoryl[3H]choline; at low amounts of cytolysin, membrane potential and intracellular content of Na+ and K+ recover within minutes. In E and Lettre cells, membrane damage is inhibited by Zn2+, by high Ca2+, or by low pH. Inhibition is reversible: addition of EGTA to Zn2+-protected E or Lettre cells (incubated in the presence of cytolysin, low Ca2+ and Zn2+) initiates leakage; removal of Zn2+ (and cytolysin and Ca2+) by washing also initiates leakage; such leakage is again sensitive to Zn2+, high Ca2+, or H+. In phospholipid bilayers, channels induced by cytolysin (at low Ca2+) are partially closed by negative voltage; Ca2+, Zn2+, or H+ promote channel closure. Channels are re-opened (only partially in the case of Zn2+) by positive voltage. From all these results it is concluded that the action of cytolysin on membranes is similar to that of other pore-forming agents: damage does not necessarily lead to lysis of nucleated cells, and can be prevented by Ca2+, Zn2+, or H+.     

Fractionation of the nucleus by divalent cations. Isolation of nuclear membranes

0.3–0.5 M MgCl₂ was used to disassemble nuclei and to isolate by a single centrifugation in less than 3 hr a nuclear envelope fraction in 55–60% yield as assessed by phospholipid recovery. Its gross chemical composition was determined and its morphology was studied electron microscopically by sectioning, freeze etching, and negative staining procedures.     

The inhibition of RNA polymerase by daunomycin

The effect of daunomycin on the in vitro activity of Escherichia coli DNA-dependent RNA polymerase has been studied under a variety of experimental conditions. The inhibition of RNA synthesis by this DNA-binding antibiotic is overcome by an increase in the DNA concentration but is unaffected by an increase in the concentration of the RNA polymerase. It is concluded that, under conditions used, the inhibition is predominantly due to the interaction of the drug with the template DNA. At the concentration used (20 μM), daunomycin is able to inhibit RNA polymerization even after its initiation. However, the possibility remains that other steps are sensitive to daunomycin. A comparison of the effect of daunomycin on RNA synthesis using different DNAs as templates suggests that the extent of inhibition depends on base composition and on the secondary structure of the DNA. The effect of base composition on the melting temperature of antibiotic-DNA complexes is consistent with the inhibiting effect on RNA synthesis.     

Monovalent cation-induced inhibition of chlorophyll a fluorescence: Antagonism by divalent cations

Salts of monovalent cations at concentrations less than 10 mm and buffers such as tricine were found to increase spillover from Photosystem II to Photosystem I in green plant photosynthesis as measured by a decrease in chlorophyll a fluorescence at room temperature. At 77 °K, they increased the fluorescence emission at 735 nm relative to the bands at 685 and 693 nm indicating that Photosystem I was receiving a greater part of the excitation energy. Divalent cations and monovalent cations at concentrations greater than 10 mm reversed the fluorescence changes.     

alpha-Amanitin resistant RNA polymerase II from Aedes albopictus cell mutants resistant to alpha-amanitin

Spontaneous and EMS-induced alpha-amanitin-resistant Aedes albopictus cells have been isolated and characterized. Two mutant sublines, one of intermediate resistance (alpha A2) and the other highly resistant (Ama18) contained RNA polymerase II activity, the resistance of which in vitro to alpha-amanitin correlated well with the resistance of these cells in vivo. The resistance of these cells to alpha-amanitin can likely be attributed to the presence of an altered RNA polymerase II.     

Purification and characterization of RNA polymerase II resistant to alpha-amanitin from the mushroom Agaricus bisporus

The DNA-dependent RNA polymerases II or B (ribonucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from the mushroom Agaricus bisporus has been purified to apparent homogeneity. The purification procedures involve precipitation with polyethylenimine, selective elution of RNA polymerase II from the polyethylenimine precipitate, ammonium sulfate fractionation, DEAE-cellulose chromatography, CM-cellulose chromatography, and exclusion chromatography on Bio-Gel A-1.5M. With this procedure 11 mg of RNA polymerase II is recovered from 1.5 kg of mushroom tissue. RNA polymerase II from Agaricus bisporus has 12 subunits with the following molecular weights: 182,000, 140,000, 89,000, 69,000, 53,000, 41,000, 37,000, 31,000, 29,000, 25,000, 19,000, and 16,500. Purified RNA polymerase II from Agaricus bisporous was half-maximally inhibited by the mushroom toxin alpha-amanitin at a concentration of 6.5 microgram/mL (7 X 10(-6) M), which is 650-fold more resistant than mammalian RNA polymerases II. The apparent Ki for the alpha-amanitin-RNA polymerase complex was estimated to be 12 X 10(-6) M. The activity of purified RNA polymerase II from the mushroom was quite typical of other eukaryotic RNA polymerase II with regard to template preference, salt optima, and divalent metal cation optima.     

Uncompetitive inhibition of Xenopus laevis aldehyde dehydrogenase 1A1 by divalent cations

Aldehyde dehydrogenases (ALDHs) convert aldehydes into their corresponding carboxylic acids. ALDH1A1, also known as ALDH class 1 (ALDH1) or retinaldehyde dehydrogenase (RALDH1), prefers retinal to acetaldehyde as a substrate. To investigate the effects of divalent cations on the dehydrogenase activity of Xenopus laevis ALDH1A1, the formation of acetate and retinoic acid from acetaldehyde and retinal, respectively, was investigated in the presence of Ca2+, Mg2+, Mn2+ or Zn2+. All divalent cations tested inhibited the oxidation of acetaldehyde and retinal by ALDH1A1. When acetaldehyde was used as a substrate, the 50% inhibitory concentrations (IC50) were 10, 24, 35 and 220 microM for Zn2+, Mn2+, Mg2+ and Ca2+, respectively. Kinetic studies of ALDH1A1 dehydrogenase activity in the presence or absence of each cation revealed that the inhibition mode by cations was uncompetitive against acetaldehyde, retinal, and NAD+, and that their inhibitory potencies were greater against acetaldehyde than retinal. It was concluded that the divalent cations inhibited X. laevis ALDH1A1 activity in a substrate-dependent manner by affecting a step of the dehydrogenase reaction that occurred after the formation of the ternary complex of the enzyme, substrate, and coenzyme.     

Pseudomonas aeruginosa acid phosphatase. Activation by divalent cations and inhibition by aluminium ion.

In Pseudomonas aeruginosa, the effect of different cations on the acid phosphatase activity was studied in order to acquire more information related to a previously proposed mechanism, involving the coordinated action of this enzyme with phospholipase C. Although the natural substrate of this enzyme is phosphorylcholine, in order to avoid the possible interaction of its positive charge and those of the different cations with the enzyme molecule, the artificial substrate p-nitrophenylphosphate was utilized. Kinetic studies of the activation of acid phosphatase (phosphorylcholine phosphatase) mediated by divalent cations Mg2+, Zn2+ and Cu2+ revealed that all these ions bind to the enzyme in a compulsory order (ordered bireactant system). The Km values obtained for p-NPP in the presence of Mg2+, Zn2+ and Cu2+ were 1.4 mM, 1.0 mM and 3.5 mM, respectively. The KA values for the same ions were 1.25 mM, 0.05 mM and 0.03 mM, respectively. The Vmax obtained in the presence of Cu2+ was about twofold higher than that obtained in the presence of Mg2+ or Zn2+. The inhibition observed with Al3+ seems to be a multi-site inhibition. The K'app and n values, from the Hill plot, were about 0.25 mM and 4.0 mM, respectively, which were independent of the metal ion utilized as activator. It is proposed that the acid phosphatase may exert its action under physiological conditions, depending on the availability of either one of these metal ions.     

DNA-dependent RNA polymerase activity of Chinese hamster kidney cells sensitive to high concentrations of alpha-amanitin

Studies on the DNA-dependent RNA polymerase activities present in Chinese Hamster Kidney Cells have revealed an enzyme inhibited only by high concentrations of α-amanitin that corresponds to the previously described RNA polymerase C. Although primarily isolated from the cytoplasmic fraction derived from these cells, evidence is presented which strongly suggests that RNA polymerase C and the nuclear RNA polymerase III are one and the same enzyme.     

Ionic channels formed byStaphylococcus aureus alpha-toxin: Voltage-dependent inhibition by divalent and trivalent cations

Summary The interaction ofStaphylococcus aureus -toxin with planar lipid membranes results in the formation of ionic channels whose conductance can be directly measured in voltage-clamp experiments. Single-channel conductance depends linearly on the solution conductivity suggesting that the pores are filled with aqueous solution; a rough diameter of 11.4±0.4 Å can be estimated for the pore. The conductance depends asymmetrically on voltage and it is slightly anion selective at pH 7.0, which implies that the channels are asymmetrically oriented into the bilayer and that ion motion is restricted at least in a region of the pore. The pores are usually open in a KCl solution but undergo a dose- and voltage-dependent inactivation in the presence of diand trivalent cations, which is mediated by open-closed fluctuations at the single-channel level. Hill plots indicate that each channel can bind two to three inactivating cations. The inhibiting efficiency follows the sequence Zn²⁺>Tb³⁺>Ca²⁺>Mg²⁺>Ba²⁺. suggesting that carboxyl groups of the protein may be involved in the binding step. A voltage-gated inactivation mechanism is proposed which involves the binding of two polyvalent cations to the channel, one in the open and one in the closed configuration, and which can explain voltage, dose and time dependence of the inactivation.