Sensor I Threonine of the AAA+ ATPase Transcriptional Activator PspF Is Involved in Coupling Nucleotide Triphosphate Hydrolysis to the Restructuring of σ54-RNA Polymerase

Transcriptional initiation invariably involves the transition from a closed RNA polymerase (RNAP) promoter complex to a transcriptional competent open complex. Activators of the bacterialσ54-RNAP are AAA+ proteins that couple ATP hydrolysis to restructure the σ54-RNAP promoter complex. Structures of the σ54 activator PspF AAA+ domain (PspF1–275) bound to σ54 show two loop structures proximal to σ54 as follows: the σ54 contacting the GAFTGA loop 1 structure and loop 2 that classifies σ54 activators as pre-sensor 1 β-hairpin AAA+ proteins. We report activities for PspF1–275 mutated in the AAA+ conserved sensor I threonine/asparagine motif (PspF1–275T148A, PspF1–275N149A, and PspF1–275N149S) within the second region of homology. We show that sensor I asparagine plays a direct role in ATP hydrolysis. However, low hydrolysis rates are sufficient for functional output in vitro. In contrast, PspF1–275T148A has severe defects at the distinct step of σ54 promoter restructuring. This defect is not because of the failure of PspF1–275T148A to stably engage with the closed σ54 promoter, indicating (i) an important role in ATP hydrolysis-associated motions during energy coupling for remodeling and (ii) distinguishing PspF1–275T148A from PspF1–275 variants involved in signaling to the GAFTGA loop 1, which fail to stably engage with the promoter. Activities of loop 2 PspF1–275 variants are similar to those of PspF1–275T148A suggesting a functional signaling link between Thr148 and loop 2. In PspF1–275 this link relies on the conserved nucleotide state-dependent interaction between the Walker B residue Glu108 and Thr148. We propose that hydrolysis is relayed via Thr148 to loop 2 creating motions that provide mechanical force to the GAFTGA loop 1 that contacts σ54.