Conformation of fork junction DNA in a complex with Escherichia coli RNA polymerase

Escherichia coli RNA polymerase is able to bind fork junction DNA containing a conserved -10 promoter element in a sequence-specific manner, and it is believed that polymerase-fork junction DNA interaction mimics those between the enzyme and the promoter DNA in the open complex. In this report we determined the conformation of polymerase-bound fork junction DNA in solution. A series of distances between sites in the fork junction DNA in complex with polymerase were determined using luminescence and fluorescence resonance energy transfer. A series of fork junction DNAs were prepared containing the luminescent or fluorescent donor probe at the upstream or at the downstream end of the fork DNA and acceptor probes at nine positions within the fork junction DNA. The measured distances were compared with analogous distances in a model reference DNA duplex, and the observed distance differences were used to build a model of the fork junction DNA in a complex with the polymerase. The obtained model revealed an insignificant perturbation of the duplex part of the fork DNA in a complex with the polymerase whereas a sharp kink of DNA was observed at the ds/ss DNA boundary of the fork junction DNA.