Abstract
Bacterial RNA polymerase (RNAP) interacts with conserved -10 and -35 promoter elements to recognize the promoter and to form an open complex in which DNA duplex around transcription start site melts. Using model DNA constructs (fork junction DNA) that mimic DNA structure found in the open complex we observed that the consequences of mutations in -10 promoter element for RNAP binding exhibited a striking dependence on the presence or absence of a functional -35 promoter element. A role of spacer DNA (a non-conserved DNA sequence connecting -10 and -35 promoter elements) in this phenomenon was probed with a series of fork junction DNA constructs containing perturbations to the spacer DNA. In the absence of a physical connection between the -10 and -35 DNA elements, or when -10 and -35 DNA elements were connected by a long flexible non-DNA linker, the dependence of RNAP interactions with -10 element on the strength of -35 element was lost. When these DNA elements were linked by a rigid DNA duplex or by a DNA duplex containing a short single-stranded gap, the coupling between the -10 and -35 binding activities was observed. These results indicated that promoter spacer DNA played an active role in integrating the functional consequences of RNA polymerase contacts with -10 and -35 promoter element. This role likely involves physical deformation of the spacer occurring in parallel with promoter melting as shown by Fluorescence Resonance Energy Transfer (FRET) experiments with the probes incorporated into spacer DNA.