T7 RNA Polymerase Interacts with Its Promoter from One Side of the DNA Helix
Daniel K. Muller, Craig T. Martin, and Joseph E. Coleman, Biochemistry 28, 3306-3313, 1989
The interactions of T7 RNA polymerase with its promoter DNA have been previously probed in footprinting experiments with either DNase I or (methidiumpropyl-EDTA)-Fe(II) to cleave unprotected DNA [Basu, S., & Maitra, U. (1986) J. Mol. Biol. 190, 425-437. Ikeda, R. A., & Richardson, C. C. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3614-3618]. Both of these reagents have drawbacks; DNase I is a bulky reagent and so provides low resolution, and (methidiumpropyl-EDTA)-Fe(II) intercalates into DNA and is therefore biased toward cleavage of double-stranded DNA. In this study, the interaction between the polymerase and the promoter has been probed with Fe(II)-EDTA. This reagent generates reactive hydroxyl radicals free in solution, which produces a more detailed picture of the polymerase-promoter complex. Two protected regions are observed on each of the two promoter DNA strands: from position -17 to position-13 and from position -7 to position -1 on the coding strand and from position -14 to position -9 and from position -3 to position +2 on the noncoding strand. From this pattern it is clear that if recognition occurs via double-stranded B-form DNA, then the protected regions lie on one face of the DNA helix, and therefore the enzyme must interact predominantly from one side of the DNA helix. Digestion of the DNA in a polymerase-promoter complex with a single-strand-specific endonuclease shows that a small region of thenoncoding strand near position -5 is susceptible to cleavage. This position is in the middle of the region that is not protected in the Fe(II)-EDTA experiments, suggesting that the promoter is at least transiently melted in this region. Interactions between two proteolyzed forms of T7 RNA polymerase and promoter DNA have also been studied. These two modified species have previously been found to possess steady-state initiation kinetics similar to that of the native enzyme; however, both have altered processive properties [Muller, D. K., Martin, C. T., & Coleman, J. E. (1988) Biochemistry 27, 5763-5771]. It is shown that these proteolyzed species are not altered in terms of the regions of promoter DNA that they protect from cleavage by Fe(II)-EDTA. Finally, the effect of binding of the initiating nucleotide on the protection pattern has been studied. GTP, GMP, and guanosine are all efficient RNA chain initiators for T7 RNA polymerase,and the latter two allow footprinting of a single state of the polymerase-promoter complex. Contrary to previous papers, no conformational change in the enzyme is induced by binding of the initiating nucleotideas detected by footprinting with Fe(II)-EDTA.
PMID: 2545254 DOI: 10.1021/bi00434a028