Mechanism of Antiactivation at the Pseudomonas sp. Strain ADP σ N -Dependent P atzT Promoter

Abstract

ABSTRACT P atzT is an internal promoter of the atzRSTUVW operon that directs the synthesis of AtzT, AtzU, AtzV, and AtzW, components of an ABC-type cyanuric acid transport system. P atzT is σ N dependent, activated by the general nitrogen control regulator NtrC with the assistance of protein integration host factor (IHF), and repressed by the LysR-type transcriptional regulator (LTTR) AtzR. We have used a variety of in vivo and in vitro gene expression and protein-DNA interaction assays to assess the mechanisms underlying AtzR-dependent repression of P atzT . Here, we show that repression only occurs when AtzR and NtrC interact simultaneously with the P atzT promoter region, indicating that AtzR acts as an antiactivator to antagonize activation by NtrC. Furthermore, repression requires precise rotational orientation of the AtzR and NtrC binding sites, strongly suggesting protein-protein interaction between the two proteins on the promoter region. Further exploration of the antiactivation mechanism showed that although AtzR-dependent repression occurs prior to open complex formation, AtzR does not alter the oligomerization state of NtrC or inhibit NtrC ATPase activity when bound to the P atzT promoter region. Taken together, these results strongly suggest that P atzT -bound AtzR interacts with NtrC to prevent the coupling of NtrC-mediated ATP hydrolysis with the remodeling of the interactions between E-σ N and P atzT that lead to open complex formation. IMPORTANCE Here, we describe a unique mechanism by which the regulatory protein AtzR prevents the activation of the σ N -dependent promoter P atzT . Promoters of this family are always positively regulated, but there are a few examples of overlapping negative regulation. The mechanism described here is highly unconventional and involves an interaction between the repressor and activator proteins to prevent the action of the repressor protein on the RNA polymerase-promoter complex.