The T7 RNA polymerase intercalating hairpin is important for promoter opening during initiation but not for RNA displacement or transcription bubble stability during elongation

The recently described crystal structures of a T7RNAP-promoter complex and an initial transcription complex reveal a P-hairpin which inserts between t he template and nontemplate strands of the promoter [Cheetham, G. M., et al . (1999) Nature 399, 80; Cheetham, G. M., et al. (1999) Science 286, 2305]. A stacking interaction between the exposed DNA bases and a valine at the t ip of this hairpin may be especially important for stabilizing the opened p romoter during initiation. It has been suggested that this hairpin may also be important for holding the transcription bubble open during transcript e longation, and a proposed model for how the RNA exits the transcription com plex implies that this hairpin may also help displace the RNA from the temp late strand. To test these hypotheses, we have characterized both point and deletion mutants of this element. We find that these mutants exhibit reduc ed activity on linear, double-stranded templates but not on supercoiled or partially single-stranded templates. Probing of promoter-polymerase complex es, initial transcription complexes, and elongation complexes with KMnO4 an d a single-strand specific endonuclease reveals that the mutants have great ly reduced promoter unwinding activity during initiation. However, the stru cture and stability of the transcription bubble during elongation are not a ltered in the mutant enzymes, and RNA displacement activity is also normal. Thus, the T7RNAP intercalating hairpin is important, though not essential, for stabilizing the opened promoter during initiation, but is not importan t for RNA displacement or for transcription bubble structure or stability d uring elongation.