T7 RNA Polymerase-Induced Bending of Promoter DNA Is Coupled to DNA Opening

Abstract

To initiate transcription, T7 RNA polymerase (RNAP) forms a specific complex with its promoter DNA and melts several base pairs near the initiation site to form an open complex. Previous gel electrophoresis studies have indicated that the promoter DNA in the initiation complex is bent [Ujvari, A., and Martin, C. T. (2000) J. Mol. Biol. 295, 1173-1184]. Here we use fluorescence resonance energy transfer (FRET) to investigate the conformation of promoter DNA in the closed and open complexes of T7 RNAP. We have used steady state and time-resolved fluorescence approaches to measure the FRET efficiency in a doubly dye-labeled duplex promoter and in a premelted bubble promoter. Changes in the FRET efficiency and hence the DNA end-to-end distance changes are small when the duplex promoter forms a complex with T7 RNAP. On the other hand, FRET changes are relatively larger when the bubble promoter binds T7 RNAP or when initiating nucleotides are added to the duplex promoter-T7 RNAP complex. The shortening of DNA end-to-end distances is indicative of DNA bending in the bubble DNA complex and in the duplex promoter complex with the initiating nucleotides. Our results are consistent with the model in which in the absence of initiating nucleotides there is a distribution of closed and open complexes, and the promoter DNA is bent slightly by <40 degrees in the closed complex but bent more sharply by 86 degrees in the open complex. The energetics of DNA bending suggests that a significant part of the available free energy from promoter and polymerase interactions is utilized in DNA bending and/or untwisting. We propose that promoter opening occurs spontaneously upon DNA bending and/or untwisting as free energy is gained through interactions of the melted promoter with the T7 RNAP active site.