The energetics of consensus promoter opening by T7 RNA polymerase.

Abstract

The consensus 23 base-pair T7 DNA promoter is classically divided into two domains, an upstream binding domain (-17 to -5), and a downstream initiation domain (-4 to +6) relative to the transcription start site at +1. During transcription initiation, T7 RNA polymerase (T7 RNAP) melts specifically the -4 to +2/+3 (TATAGG/G) region of the duplex DNA promoter to form a pre-initiation open complex. No external energy source is used and the energy for open complex formation is derived from the free energy of specific interactions with the binding domain, particularly the specificity region (-13 to -6). Using 2-aminopurine fluorescence-based equilibrium and kinetic measurements, we have measured the binding affinities of various topologically modified DNA promoters (40 bp in length) that represent initial, final, and transition-state analogs of the promoter DNA in the T7 RNAP-DNA complex, to determine the energy of specific binding interactions, and the energy required for forming an initiation bubble. The results indicate that 16-16.5 kcal mol(-1) of free energy is made available upon T7 RNAP binding (through specificity loop) to the promoter binding domain. To melt the TATAGG/G sequence 7-8 kcal mol(-1) of free energy is utilized; this compares with approximately 6 kcal mol(-1) predicted from nearest neighbor analysis. The remaining 8.5-9.5 kcal mol(-1) of net free energy is retained for stabilization of the specific pre-initiation binary complex. Of the 7-8 kcal mol(-1) energy that is used to generate the pre-initiation DNA bubble in the open complex, we estimate that one half (3.5-4 kcal mol(-1)) is utilized for nucleation/deformation process (through bending, untwisting, etc.) in the melting region (-4 to -1 TATA) of the initiation domain (-4 to +6), and appears to be independent of the nucleation site within this region. The other half is utilized in unpairing the +1 to +2/+3 GG/G sequence for initiation. The interactions of T7 RNAP with a 20-bp non-specific DNA on the other hand are very weak (DeltaG<-5k cal mol(-1)), which is not sufficient to melt and stabilize an open complex of a non-specific DNA.