Fine structure of the promoter-sigma region 1.2 interaction

We recently proposed that a nontemplate strand base in the discriminator region of bacterial promoters, the region between the -10 element and the transcription start site, makes sequence-specific contacts to region 1.2 of the sigma subunit of Escherichia coli RNA polymerase (RNAP). Because rRNA promoters contain sequences within the discriminator region that are suboptimal for interaction with sigma1.2, these promoters have the kinetic properties required for regulation by the RNAP-binding factors DksA and ppGpp. Here, we use zero-length cross-linking and mutational, kinetic, and footprinting studies to map RNAP interactions with the nontemplate strand bases at the junction of the -10 element and the discriminator region in an unregulated rRNA promoter variant and in the lambdaP(R) promoter. Our studies indicate that nontemplate strand bases adjacent to the -10 element bind within a 9-aa interval in sigma1.2 (residues 99-107). We also demonstrate that the downstream-most base on the nontemplate strand of the -10 hexamer cross-links to sigma region 2, and not to sigma1.2. Our results refine models of RNAP-DNA interactions in the promoter complex that are crucial for regulation of transcription initiation.