Distinct Interaction Mechanism of RNAP and ResD and Distal Subsites for Transcription Activation of Nitrite Reductase in Bacillus subtilis ψ

Abstract

The ResD-ResE signal transduction system plays a pivotal role in anaerobic nitrate respiration in Bacillus subtilis . The nasD operon encoding nitrite reductase is essential for nitrate respiration and is tightly controlled by the ResD response regulator. To understand the mechanism of ResD-dependent transcription activation of the nasD operon, we explored ResD-RNA polymerase (RNAP), ResD-DNA, and RNAP-DNA interactions required for nasD transcription. Full transcriptional activation requires the upstream promoter region where five molecules of ResD bind. The distal ResD-binding subsite at −87 to −84 partially overlaps a sequence similar to the consensus distal subsite of the upstream (UP) element with which the Escherichia coli C-terminal domain of the α subunit (αCTD) of RNAP interacts to stimulate transcription. We propose that interaction between αCTD and ResD at the promoter-distal site is essential for stimulating nasD transcription. Although nasD has an extended −10 promoter, it lacks a reasonable −35 element. Genetic analysis and structural simulations predicted that the absence of the −35 element might be compensated by interactions between σ A and αCTD, and between αCTD and ResD at the promoter-proximal ResD-binding subsite. Thus, our work suggested that ResD likely participates in nasD transcription activation by binding to two αCTD subunits at the proximal and distal promoter sites, representing a unique configuration for transcription activation. IMPORTANCE A significant number of ResD-controlled genes have been identified and transcription regulatory pathways in which ResD participates have emerged. Nevertheless, the mechanism of how ResD activates transcription of different genes in a nucleotide sequence-specific manner has been less explored. This study suggested that among the five ResD-binding subsites in the promoter of the nasD operon, the promoter-proximal and -distal ResD-binding subsites play important roles in nasD activation by adapting different modes of protein-protein and protein-DNA interactions. The finding of a new-type of protein-promoter architecture provides insight into the understanding of transcription activation mechanisms controlled by transcription factors including the ubiquitous response regulators of two-component regulatory systems particularly in Gram-positive bacteria.