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.     

Effects of estradiol on uterine ribonucleic acid metabolism. Assessment of transfer ribonucleic acid methylation.

Immature rats treated with estradiol for selected periods of time demonstrated both increased methylation of uterine transfer ribonucleic acid (tRNA) and methylase activities. Whereas the former parameter was assessed by incubating whole uteri with [methyl-14C]methionine and measuring the incorporation of isotope into the tRNA, methylase activity was obtained by measuring the rate of incorporation of methyl groups from S-adenosyl[methyl-14C]methionine into heterologous tRNA (Escherichia coli B) in the presence of uterine cytosol preparations (100,000g supernatants). Although increased methylation of tRNA during the estrogen response was demonstrated, additional studies indicated that these results were largely attributable to an increased rate of synthesis of tRNA rather than gross changes in either the type or amount of methylated constituents present. Evidence in this regard included the inability of estrogen treatment of alter significantly the (a) resulting patterns of methyl-14C-methylated constituents of uterine tRNA, (b) the extent ot which [2-14C]guanine residues, incorporated into tRNA, become methylated, (c) the extent of methylation of precursor tRNA in the absence of tRNA synthesis, and (d) the types of methylase activities expressed in vitro.     

Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Cognate transfer ribonucleic acid as a ligand.

The use of tRNA affinity columns for the purification of aminoacyl-tRNA synthetases was investigated. A purification method for valyl-tRNA synthetase from Bacillus stearothermophilus is described that uses two affinity columns, one containing the pure cognate tRNA, and the other containing all tRNA species except the cognate tRNA. A method for the rapid preparation of the two columns was developed, which does not require prior isolation of cognate tRNA but makes use of the ability of the target synthetase to select its cognate tRNA. The usefulness of tRNA columns is compared with that of affinity columns derived from the aminoalkyladenylate reported in the preceding paper [Clarke & Knowles (1977) Biochem J. 167, 405-417].     

Inhibition of Histoplasma capsulatum ribonucleic acid polymerases by homologous and heterologous ribonucleic acid.

The ribonucleic acid (RNA) polymerases from the yeast phase of Histoplasma capsulatum are differentially sensitive to RNA isolated from the yeast and mycelial phases of this fungus and from Escherichia coli. Low-molecular-weight RNA from H. capsulatum was the most effective inhibitor.     

Conservation of transfer ribonucleic acid and 5S ribonucleic acid cistrons in Enterobacteriaceae.

The genes for tranfer ribonucleic acid (tDNA) and 5S ribonucleic acid (5SDNA) were isolated from the total deoxyribonucleic acid (DNA) of Escherichia coli. The relatedness of tDNA and 5S from E. coli and other species of Enterobacteriaceae was determined by reassociation of the isolated genes labeled with 32PO4 to unlabeled, unfractionated DNA. Double-stranded DNA was separated from unreacted DNA by hydroxyapatite chromatography. Thermal elution profiles were done to determine the amount of unpaired bases present in related DNA sequences. Relative to total DNA, both 5S DNA and tDNA were highly conserved throughout the Enterobacteriaceae, including the genera Yersinia and Proteus.     

Inhibition of leucyl-transfer ribonucleic acid synthetasymol.

The bacteriostatic effect of low concentrations of the antibiotic granaticin on Bacillus subtilis is relieved by the addition leucine to the growth medium. In cells treated with granaticin, aminoacylation of leucine tRNA is specifically decreased, but the content of free leucine is not. It is concluded that granaticin interferes with the charging process of leucine tRNA in B. subtilis leading to leucine auxotrophy.     

Regulation of the biosynthesis of aminoacyl-transfer ribonucleic acid synthetases and of transfer ribonucleic acid in Escherichia coli. V. Mutants with increased levels of valyl-transfer ribonucleic acid synthetase.

Spontaneous revertants of a temperature-sensitive Escherichia coli strain harboring a thermolabile valyl-transfer ribonucleic acid (tRNA) synthetase were selected for growth at 40 degrees C. Of these, a large number still contain the thermolabile valyl-tRNA synthetase. Three of these revertants contained an increased level of the thermolabile enzyme. The genetic locus, valX, responsible for the enzyme overproduction, is adjacent to the structural gene, valS, of valyl-tRNA synthetase. Determination (by radioimmunoassay) of the turnover rates of valyl-tRNA synthetase showed that the increased level of valyl-tRNA synthetase is due to new enzyme synthesis rather than decreased rates of protein degradation.