Initial steps in the large-scale purification of Escherichia coli deoxyribonucleic acid-dependent ribonucleic acid polymerase

Histones from exponential and stationary-phase mouse L-cells were quantitated after acrylamide gel electrophoresis in order to investigate cell cycle-dependent changes in the mode of binding of the various fractions in chromatin. By introducing various concentrations of citrate and divalent cations in the medium used for cell lysis and isolation of nuclei prior to histone extraction it was possible to demonstrate that certain histone fractions are preferentially retained in either exponential or stationary-phase nuclei. Differential retention of lysine-rich F₁ was most evident when the lysing medium contained 1 mm Mg²⁺ and Ca²⁺ and 5 mm citrate (pH 2.75). In these conditions twice as much F₁ is retained in stationary as in exponential nuclei. Differential retention of arginine-rich histones was most evident when the lysing medium contained 10 mm Mg²⁺ and Ca²⁺ and no citrate. In these conditions more F_{2a 1} is retained in exponential than in stationary nuclei while the opposite is true for F₃. However, the total amount of arginine-rich fractions (F_{2a 1} + F₃) retained was found to be the same in both cell phases. The results are discussed in relation to known structural features of the histones.     

Kinetics and template-dependency of ribonucleic acid synthesis by bacterial ribonucleic acid polymerase.

The rate of RNA synthesis catalysed by DNA-dependent RNA polymerase shows a Michealis-Menten-type saturation curve with increasing template concentration. However, the apparent Km is proportional to enzyme concentration, indicating that the reaction does not obey a simple kinetic scheme. The action of inhibitors also indicates a more complex interaction between the enzyme and the DNA template; many inhibitors of RNA synthesis either decrease Vmax. without affecting Km, or increase Km without affecting Vmax. All of these observations can be accounted for quantitatively by a reaction pathway in which the non-specific binding sites of the viral DNA template inhibit competitively the binding of the enzyme to the initiation sites. In terms of this pathway the two classes of inhibitors of RNA synthesis must then act predominantly either on the rate of elongation or on the availability of the binding sites respectively.     

Affinity labeling of the 3'-OH terminal binding site of the ribonucleic acid chain on deoxyribonucleic acid dependent ribonucleic acid polymerase from Escherichia coli.

Nucleoside triphosphates modified at the 3'-OH are chain terminators for RNA polymerase. They form inactive ternary complexes with the enzyme, poly(dT), and oligoadenylate, the stabilities of which depend upon the length of the oligonucleotide. Employing [5'-32P]p(Ap)10A, together with the reactive analogues 3'-(bromoacetamide)-3'-deoxyadenosine triphosphate or 3'-(isothiocyanato)-2',3'-dideoxyadenosine triphosphate, as well as 3'-amino-3'-deoxyadenosine triphosphate, followed by cross-linking with glyoxal, we labeled RNA polymerase primarily at the beta' subunit. The latter therefore appears to contain at least in part the 3'-OH terminus of the nascent RNA chain when the enzyme is in the form of the ternary complex.     

The inhibition of ribonucleic acid polymerase by acridines

1. The aminoacridines, proflavine (3,6-diaminoacridine) and 9-aminoacridine, and a hydrogenated derivative, 9-amino-1,2,3,4-tetrahydroacridine, were shown to inhibit in vitro the DNA-primed RNA polymerase of Escherichia coli. The inhibition is strong with both proflavine and 9-aminoacridine, but weak with 9-amino-1,2,3,4-tetrahydroacridine. 2. The extent to which the three acridines bind to calf-thymus DNA in the enzyme medium was studied spectrophotometrically. The extent of binding decreases in the order: proflavine, 9-aminoacridine, 9-amino-1,2,3,4-tetrahydroacridine. Some evidence was also obtained for interaction between the nucleoside triphosphate substrates and proflavine or 9-aminoacridine; no such interaction was detectable with 9-amino-1,2,3,4-tetrahydroacridine. 3. Although the amount of acridine bound to DNA increases with increasing inhibition, a stage is reached where an increase in acridine concentration still causes an increase in inhibition, with practically no increase in the amount bound to DNA. 4. Plots of reciprocal rates against the reciprocal of DNA concentration were linear and had a common intercept when proflavine or 9-aminoacridine was present. Similar relations were obtained when the reciprocal concentration of nucleoside triphosphates was plotted. The observations are interpreted kinetically in terms of a competitive inhibition of the enzyme by proflavine or 9-aminoacridine and of a kinetic role for the DNA analogous to ;activation'. 5. This suggests that inhibitory acridine molecules can occupy the sites on the RNA polymerase that are specific for binding the nucleoside triphosphate substrate or the bases of the DNA, when these become accessible during the copying process.     

Specific photoaffinity labeling of the cAMP surface receptor in Dictyostelium discoideum

The recent observation that ammonium sulfate stabilizes cell-surface [3H]cyclic AMP binding in Dictyostelium discoideum (Van Haastert, P., and Kien, E. (1983) J. Biol. Chem. 258, 9636-9642) led us to attempt to identify the surface cAMP receptor by photoaffinity labeling with 8-azido-[32P]cAMP using this stabilization technique. 8-azido-[32P]cAMP specifically labeled a polypeptide which migrates as a closely spaced doublet (Mr = 40,000 to 43,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Greater than 60% of the labeled polypeptide was found associated with membranes. This protein was distinguished from the cytosolic regulatory subunit of the cAMP-dependent protein kinase (Mr = 41,000) by differences in developmental regulation, specificity, and subcellular localization. No kinase regulatory subunit was detected in membranes by western blot analysis. Our preliminary observations show that labeling of this doublet correlates closely with cAMP-binding activity, suggesting that it is the surface receptor which mediates chemotaxis and cAMP signaling.