Interactions of T7 RNA Polymerase with T7 Late Promoters Measured by Footprinting with Methidiumpropyl-EDTA-Iron(II)

Samuel I. Gunderson, Kenneth A. Chapman, Richard R. Burgess

Research output: Contribution to journalArticle

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Abstract

The interactions of T7 RNA polymerase with T7 late promoters were studied by using quantitative footprinting with methidiumpropyl-EDTA•Fe(II) [MPE-Fe(II)] as the DNA cleaving agent. Class II and class III T7 promoters have a highly conserved 23 base pair sequence from -17 to +6. Among class III promoters the -22 to -18 region is also highly conserved. For a class II promoter, T7 RNA polymerase protects the -17 to -4 region from MPE-Fe(II) cleavage; when GTP is present, protection extends from -17 to +5 (noncoding strand). For a class III promoter, protection extends from -20 to -4 and in the presence of GTP from -20 to +5 (noncoding strand). The protected regions for the coding strands of both promoters were nearly identical with that seen for the noncoding strands. The binding constant for the class III promoter is (4 ± 1.5) X 107M−1 and in the presence of GTP increases to (10 ± 1.7) X 107M−1. These binding constants are about 1000 and 200 times greater, respectively, than values reported previously [Ikeda, R. A., & Richardson, C. C. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3614–3618]. The differences in binding constants are probably due to tRNA and high salt used in those earlier experiments. Both tRNA and high salt (>50 mM NaCl and >10 mM MgCl2) inhibit the binding of the polymerase to the promoter. Optimal binding conditions occur at 2–5 mM MgCl2 and 0–10 mM NaCl. Finally, the binding constant between T7 RNA polymerase and nonpromoter DNA sites was determined to be (2.1 ± 1.1) X 104M−1.

Original languageEnglish (US)
Pages (from-to)1539-1546
Number of pages8
JournalBiochemistry
Volume26
Issue number6
DOIs
StatePublished - Jan 1 1987
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry

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