Abstract:
: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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Strauss HS,Burgess RR,Record MT Jrdoi
10.1021/bi00556a014subject
Has Abstractpub_date
1980-07-22 00:00:00pages
3496-504issue
15eissn
0006-2960issn
1520-4995journal_volume
19pub_type
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