Genetic Evidence for a Novel Interaction between Transcriptional Activator SoxS and Region 4 of the σ70 Subunit of RNA Polymerase at Class II SoxS-Dependent Promoters in Escherichia coli

Escherichia coli SoxS activates transcription of the genes of the soxRS regulon, which provide the cellʹs defense against oxidative stress. In response to this stress, SoxS is synthesized de novo. Because the DNA binding site of SoxS is highly degenerate, SoxS efficiently activates transcription by the mechanism of prerecruitment. In prerecruitment, newly synthesized SoxS first forms binary complexes with RNA polymerase. These complexes then scan the chromosome for class I and II SoxS-dependent promoters, using the specific DNA-recognition properties of SoxS and σ70 to distinguish SoxS-dependent promoters from the vast excess of sequence-equivalent soxboxes that do not reside in promoters. Previously, we determined that SoxS interacts with RNA polymerase in two ways: by making protein–protein interactions with the DNA-binding determinant of the α subunit and by interacting with σ70 region 4 (σ70 R4) both “on-DNA” and “off-DNA.” Here, we address the question of how SoxS and σ70 R4 coexist at class II promoters, where the binding site for SoxS either partially or completely overlaps the − 35 region of the promoter, which is usually bound by σ70 R4. To do so, we created a tri-alanine scanning library that covers all of σ70 R4. We determined that interactions between σ70 R4 and the DNA in the promoterʹs − 35 region are required for activation of class I promoters, where the binding site lies upstream of the − 35 hexamer, but they are not required at class II promoters. In contrast, specific three-amino-acid stretches are required for activation of class I (lac) and class II (galP1) cyclic AMP receptor protein-dependent promoters. We conclude from these data that SoxS and σ70 R4 interact with each other in a novel way at class II SoxS-dependent promoters such that the two proteins do not accommodate one another in the − 35 region but instead SoxS binding there occludes the binding of σ70 R4.