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Bacterial enhancer binding proteins (bEBPs) are transcription activators that belong to the AAA(+) protein family. They form higher-order self-assemblies to regulate transcription initiation at stress response and pathogenic promoters. The precise mechanism by which these ATPases utilize ATP binding and hydrolysis energy to remodel their substrates remains unclear. Here we employed mass spectrometry of intact complexes to investigate subunit dynamics and nucleotide occupancy of the AAA(+) domain of one well-studied bEBP in complex with its substrate, the σ(54) subunit of RNA polymerase. Our results demonstrate that the free AAA(+) domain undergoes significant changes in oligomeric states and nucleotide occupancy upon σ(54) binding. Such changes likely correlate with one transition state of ATP and are associated with an open spiral ring formation that is vital for asymmetric subunit function and interface communication. We confirmed that the asymmetric subunit functionality persists for open promoter complex formation using single-chain forms of bEBP lacking the full complement of intact ATP hydrolysis sites. Outcomes reconcile low- and high-resolution structures and yield a partial sequential ATP hydrolysis model for bEBPs.

Original publication

DOI

10.1016/j.jmb.2013.08.018

Type

Journal article

Journal

J Mol Biol

Publication Date

09/01/2014

Volume

426

Pages

71 - 83

Keywords

AAA(+) protein, CID, MS, PspF, WT, bEBP, bacterial enhancer binding protein, collision-induced dissociation, enhancer binding protein, mass spectrometry, nanoESI-MS, nanoelectrospray ionization mass spectrometry, native mass spectrometry, sigma 54, wild type, Adenosine Triphosphatases, Mass Spectrometry, Models, Biological, Nucleotides, Protein Subunits, RNA Polymerase Sigma 54, Trans-Activators, Transcription, Genetic