Binding of Escherichia coli ribonucleic acid polymerase holoenzyme to a bacteriophage T7 promoter-containing fragment: selectivity exists over a wide range of solution conditions.
The selectivity of binding of Escherichia coli RNA polymerase holoenzyme to a promoter-containing fragment of T7 DNA has been 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. We find that 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 0-37 degrees C. By contrast, the nonspecific interaction of polymerase with the nonpromoter fragment is known to be relatively insensitive to pH and temperature, though a strong function of salt concentration [deHaseth, O. L., Lohman, T. M., Burgess, R. R., & Record, M. T., Jr. (1978) Biochemistry 17, 1612-1622]. Selectivity of binding of RNA polymerase in our assay is demonstrated by a greater fractional retention of the promoter-containing fragment than of the nonpromoter fragment on the filter. We observe selective binding over the temperature range from 0 to 37 degrees 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. Quantitative treatment of these binding data [Strauss, H. S., Burgess, R. R., & Record, M. t., Jr. (1980) Biochemistry (second paper of four in this issue)] confirms these conclusions and shows that selectivity is a function of ion concentration as well.
D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009154:Mutation; D009876:Operon; D011485:Protein Binding; D012964:Sodium / Q000494:pharmacology; D013604:T-Phages / Q000201:enzymology*
D004279:DNA, Viral; D012964:Sodium; D012321:DNA-Directed RNA Polymerases; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases; D008274:Magnesium