Proteins that belong to the AAA (ATPases associated with various cellular activities) superfamily of mechanochemical enzymes are versatile and control a wide array of cellular functions. Many AAA proteins share the common property of self-association into oligomeric structures and use nucleotide binding and hydrolysis to regulate their biological output. The Escherichia coli transcription activator PspF (phage shock protein F) is a member of the σ54-dependent transcriptional activators that belong to the AAA protein family. Nucleotide interactions condition the functional state of PspF, enabling it to self-associate and interact with its target, the σ54–RNAP (RNA polymerase) closed complex. The self-association determinants within the AAA domain of σ54-dependent activators remain poorly characterized. In the present study, we have used a fragment of the AAA domain of PspF as a probe to study the nucleotide-conditioned self-association of PspF. Results show that the PspF fragment acts in trans to inhibit specifically self-association of PspF. The PspF fragment prevented efficient binding of nucleotides to PspF, consistent with the observation that the site for nucleotide interactions within an oligomer of AAA proteins is created between two protomers. Using proximity-based footprinting and cross-linking techniques, we demonstrate that the sequences represented in this fragment are close to one protomer–protomer interface within a PspF oligomer. As the sequences represented in this PspF fragment also contain a highly conserved motif that interacts with the σ54–RNAP closed complex, we suggest that PspF may be organized to link nucleotide interactions and self-association to σ54–RNAP binding and transcription activation.
sigma54-dependent transcription activator phage shock protein F of Escherichia coli: a fragmentation approach to identify sequences that contribute to self-association
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Patricia BORDES, Siva R. WIGNESHWERARAJ, Xiaodong ZHANG, Martin BUCK; sigma54-dependent transcription activator phage shock protein F of Escherichia coli: a fragmentation approach to identify sequences that contribute to self-association. Biochem J 15 March 2004; 378 (3): 735–744. doi: https://doi.org/10.1042/bj20031464
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