Binding of Escherichia coli ribonucleic acid polymerase holoenzyme to a bacteriophage T7 promoter-containing fragment: evaluation of promoter binding constants as a function of solution conditions

Abstract

Thermodynamic and molecular information was obtained on the specific complexes formed between E. coli RNA polymerase holoenzyme and a restriction fragment of T7 D111 DNA carrying the A1 and D promoters. Specific binding was observed at 0.degree. and 37.degree. C over a wide range of pH values and ion concentrations. The specific complexes formed at these 2 temperatures may correspond to the closed and open promoter complexes. Promoter binding constants KobsdRP are obtained from competition filter binding data by using a statistical analysis and previously determined values of the nonspecific holoenzyme-DNA binding constant KobsdRD. The magnitudes of KobsdRP at 0.degree. and 37.degree. C and the dependences of these binding constants on pH and ion concentrations indicate that under physiological ionic conditions, both the 0.degree. and the 37.degree. C complexes are stabilized to a large extent by the formation of ionic interactions and the accompanying release of counterions and that 1 or 2 protonation events (pK .apprx. 7.4) are required for complex formation in both cases. The 0.degree. and 37.degree. C complexes differ in their sensitivity to ion concentrations and in the magnitude of KobsdRP. The 2 complexes are probably distinct. (More counterion release accompanies formation of the 37.degree. complex). Comparisons of the 2 complexes with each other and with nonspecific holoenzyme-DNA complexes are drawn from the binding data. The equilibrium selectivity ratio (KobsdRP/KobsdRD) was a sensitive function of temperature and ionic conditions. Selectivity of holoenzyme for promoter sites on the promoter-containing fragment was higher at 37.degree. C than at 0.degree. C under the conditions investigated. Selectivity at either temperature increased by reducing the pH (in the range 6.1-8.6). At 37.degree. C, selectivity increased by reducing the salt concentration. Under approximately physiological conditions (0.2 M NaCl and 0.003 M MgCl2, pH 7.4, 37.degree. C), the equilibrium selectivity ratio was of the order of magnitude 104.