Binding of Escherichia coli ribonucleic acid polymerase holoenzyme to a bacteriophage T7 promoter-containing fragment: selectivity exists over a wide range of solution conditions

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Abstract
The selectivity of binding of E. coli RNA polymerase holoenzyme to a promoter-containing fragment of T7 DNA was investigated over a range of solution conditions by using a double-label nitrocellulose filter binding assay. A 32P-labeled HaeIII restriction fragment of T7 D111 DNA containing the A1 and D promoters for the E. coli enzyme and a 3H-labeled nonpromoter HaeIII fragment of comparable size were incubated with .sigma.-saturated holoenzyme and filtered through a nitrocellulose membrane filter. The extent of binding of polymerase to the promoter-containing fragment decreases dramatically with increasing salt concentrations and with increasing pH and increases moderately with increasing temperature in the range 0.degree.-37.degree. C. The nonspecific interaction of polymerase with the nonpromoter fragment is relatively insensitive to pH and temperature, though a strong function of salt concentration. Selectivity of binding of RNA polymerase in this assay was demonstrated by a greater fractional retention of the promoter-containing fragment than of the nonpromoter fragment on the filter. Selective binding was observed over the temperature range 0.degree.-37.degree. C near neutral pH and over a wide range of Na+ concentrations, in the presence or absence of Mg2+. Because of the different dependences of promoter and nonpromoter binding on pH and temperature, the extent of selectivity increases with increasing temperature and decreases with increasing pH.