Effects of flanking DNA and a transcriptional activator on the histone acetyltransferase activity of the SAGA complex

Though many of the proteins and protein complexes involved in eukaryotic gene expression have been identified, mechanistic knowledge about how the proteins interact with and influence one another to promote gene expression remains limited. In this work, we have started to tease out the effects of an activator protein and the chromatin environment on the HAT activity of the SAGA complex. In contrast to the prevailing model of stimulation in which activator was believed to increase the binding affinity of SAGA for chromatin, we observed that activator increases the turnover rate of acetylation by SAGA on chromatin substrates. We have determined that this stimulation is dependent on (1) the binding of activator to its consensus sequence in the flanking DNA of a target nucleosome, (2) the ability of SAGA to compete for nucleosome binding with non-specific activator binding, and (3) the interaction of activator with the Tra1 subunit of SAGA. We have further analyzed the effects of activator on another HAT complex, the poorly-characterized ADA complex. Additionally, we have characterized the influence of nucleosome flanking DNA on the HAT activity of SAGA; the presence of flanking DNA stimulates HAT activity by increasing both binding affinity and turnover rate. Altogether, the work presented here suggests a model where SAGA binds to and acetylates nucleosomes inefficiently until it interacts with DNA-bound activators or exposed nucleosome flanking DNA near gene promoters. The interaction between activator or DNA and SAGA stimulates its HAT activity, thereby generating localized regions of hyperacetylation at gene promoters

Hydrolysis of Phenyl Furyl Ketimine - The Relative Negativity Effect

Phenyl furyl ketimine hydrochloride has been prepared and the velocity of its hydrolysis to the corresponding ketone measured. The velocity constant has been found to be of the order of l0xl0-3 measured at 25°C. The constant for diphenyl ketimine hydrochloride is 5.5x103measured at 0° or about 50x10-3 when calculated to 25 °. In view of the generally considered more highly negative character of the furyl radical over the phenyl, this result is in line with an observed rule that the ketimine salts are more resistant to hydrolysis the more negative the radicals attached to the carbimino group. The hydrolytic velocities of these and other ketimines already studied are compared on the basis of Kharasch\u27s table of relative negativities of aromatic radicals

A Study of the Hoesch-Houben Synthesis in the Preparation of Aromatic Ketimines and Hydroxy-Phenyl Iminoesters

This synthesis involves the condensation of a phenol with a nitrile (aliphatic or aromatic). It is effected by the passage of dry hydrogen chloride through their solution in anhydrous ether, with or without the addition of dry zinc chloride. Anhydrous aluminum chloride was substituted for zinc chloride in some cases

The Preparation and Properties of Furyl Phenyl Ketimine

This ketimine has been prepared through the condensation of furonitrile with magnesium phenyl bromide, followed by treatment with ice and ammonium chloride at about -15 degrees C. The hydrochloride, a white solid, is rather quickly hydrolyzed to the corresponding ketone, benzoyl furane. The velocity of this hydrolysis and the basic strength of the free ketimine base are determined

Effects of Hydrogen Ion Concentration upon the Hydrolysis Rates of Ketimines (Abstract)

Measurements have been made upon the hydrolysis rates of ketimine hydrochlorides to obtain information concerning the relation between their varied structures and stability toward hydrolysis1. The hydrogen ion concentration might appear as a catalytic factor in this hydrolysis2. It would thus seem necessary to determine these hydrolysis rates at the same hydrogen ion concentration if this factor has a marked effect. Different ketimine hydrochlorides alone in water produce, in the dilute solutions usually employed, pH values ranging initially from about a.6 to 5.3 and increasing in each case toward the pH of the equivalent ammonium chloride formed as the result of hydrolysis. Certain measurements on the hydrolysis rates of ketimine hydrochlorides of widely different initial pH values in water, to which an equivalent of hydrochloric acid was added, showed that their speed was actually lowered by this hydrogen ion increase although not enough to affect the general order of the rate

Preparation and Stability of Diphenyl Amine Hydronitrate

In connection with certain proposed studies the question of the preparation of diphenyl amine hydronitrate, or diphenyl ammonium nitrate, arose. A search of the literature revealed no record of this substance. The nearest equivalent reported was the perchlorate salt of diphenyl amine (Ber. 43, 1085-86. 1910). This compound was obtained upon treatment of a carbon tetrachloride solution of the amine with 70% perchloric acid added dropwise

The Strengths of Phenolic Ketimines and Their Methyl Ethers as Bases

The ionization constants of the monohydroxy-diphenyl ketimines and their methyl ethers are calculated from measurements of the hydrogen ion concentration of aqueous solutions of their hydrochlorides through the use of the quinhydrone electrode. This data has been collected as a part of the information expected to throw light on the varied stability toward hydrolysis shown by different ketimines

The Synthesis of 4-Hydroxy and 2-Hydroxy-Diphenyl Ketimines

These syntheses were carried out through the action of the corresponding hydroxy-nitrile upon magnesium phenyl bromide in an anhydrous ether medium. Over two equivalents of the Grignard reagent were employed, one to react with the phenol group and the other with the nitrile. The addition compound was decomposed with water and ammonium chloride at about -15 degrees C. The ketimine extracted with ether was precipitated as the hydrochloride by the addition of dry hydrogen chloride. The hydroxynitriles were prepared from the corresponding hydroxy-aldehydes by the action of acetic anhydride on their oximes. Some modifications were worked out in the preparation of the oximes and the nitriles