Patricia Bordes

Mechanochemical ATPases and transcriptional activation

Transcriptional activator proteins that act upon the σ54‐containing form of the bacterial RNA polymerase belong to the extensive AAA+ superfamily of ATPases, members of which are found in all three kingdoms of life and function in diverse cellular processes, often via chaperone‐like activities. Formation and collapse of the transition state of ATP for hydrolysis appears to engender the interaction of the activator proteins with σ54 and leads to the protein structural transitions needed for RNA polymerase to isomerize and engage with the DNA template strand. The common oligomeric structures of AAA+ proteins and the crea‐tion of the active site for ATP hydrolysis between protomers suggest that the critical changes in protomer structure required for productive interactions with σ54‐holoenzyme occur as a consequence of sensing the state of the γ‐phosphate of ATP. Depending upon the form of nucleotide bound, different functional states of the activator are created that have distinct substrate and chaperone‐like binding activ‐ities. In particular, interprotomer ATP interactions rely upon the use of an arginine finger, a situation reminiscent of GTPase‐activating proteins.

The ATP hydrolyzing transcription activator phage shock protein F of Escherichia coli: Identifying a surface that binds σ54

Members of the protein family called ATPases associated with various cellular activities (AAA+) play a crucial role in transforming chemical energy into biological events. AAA+ proteins are complex molecular machines and typically form ring-shaped oligomeric complexes that are crucial for ATPase activity and mechanism of action. The Escherichia coli transcription activator phage shock protein F (PspF) is an AAA+ mechanochemical enzyme that functions to sense and relay the energy derived from nucleoside triphosphate hydrolysis to catalyze transcription by the σ54-RNA polymerase. Closed promoter complexes formed by the σ54-RNA polymerase are substrates for the action of PspF. By using a protein fragmentation approach, we identify here at least one σ54-binding surface in the PspF AAA+ domain. Results suggest that ATP hydrolysis by PspF is coupled to the exposure of at least one σ54-binding surface. This nucleotide hydrolysis-dependent presentation of a substrate binding surface can explain why complexes that form between σ54 and PspF are transient and could be part of a mechanism used generally by other AAA+ proteins to regulate activity.

Molecular Determinants for PspA-Mediated Repression of the AAA Transcriptional Activator PspF

The Escherichia coli phage shock protein system (pspABCDE operon and pspG gene) is induced by numerous stresses related to the membrane integrity state. Transcription of the psp genes requires the RNA polymerase containing the sigma(54) subunit and the AAA transcriptional activator PspF. PspF belongs to an atypical class of sigma(54) AAA activators in that it lacks an N-terminal regulatory domain and is instead negatively regulated by another regulatory protein, PspA. PspA therefore represses its own expression. The PspA protein is distributed between the cytoplasm and the inner membrane fraction. In addition to its transcriptional inhibitory role, PspA assists maintenance of the proton motive force and protein export. Several lines of in vitro evidence indicate that PspA-PspF interactions inhibit the ATPase activity of PspF, resulting in the inhibition of PspF-dependent gene expression. In this study, we characterize sequences within PspA and PspF crucial for the negative effect of PspA upon PspF. Using a protein fragmentation approach, we show that the integrity of the three putative N-terminal alpha-helical domains of PspA is crucial for the role of PspA as a negative regulator of PspF. A bacterial two-hybrid system allowed us to provide clear evidence for an interaction in E. coli between PspA and PspF in vivo, which strongly suggests that PspA-directed inhibition of PspF occurs via an inhibitory complex. Finally, we identify a single PspF residue that is a binding determinant for PspA.

Enhancer-dependent transcription by bacterial RNA polymerase: The β subunit downstream lobe is used by σ54 during open promoter complex formation

Publisher Summary This chapter describes experimental systems used in probing the function of the β subunit downstream lobe in the context of RNAP containing the major variant σ subunit, the enhancer-dependent σ factor, σ 54 . The protocols described in the chapter provide a simple step-by-step guide to obtain a relatively pure preparation of proteins required to study enhancer-dependent transcription. The experimental assays are designed to measure in vitro reconstitution of the σ 54 –RNAP and to assess the extent of DNA opening by the σ 54 –RNAP in response to activation. These assays are used in conjunction with potassium permanganate probing of wild-type and β(Δ186-433)E σ 54 –RNAP promoter complexes to show that RNAP β subunit residues 186–433 are used commonly by the enhancer-independent E σ 70 and enhancer-dependent E σ 54 for open promoter complex formation en route to transcription initiation.

The Second Paradigm for Activation of Transcription

Publisher Summary Gene transcription is central to the development, differentiation, and adaptation of cells. Control of transcription requires the interplay of signaling pathways with the molecular machinery of transcription, the DNA-dependent RNA polymerase (RNAP) enzyme, regulatory proteins that act upon it, and the nucleic acid that is transcribed. The genetic tractability of bacteria, in particular Escherichia coli and Bacillus subtilis, and yeast has allowed rapid progress in elucidating the types of strategy used for the control of gene expression at the level of transcription. The RNAP is evolutionarily conserved in sequence, structure, and function from bacteria to humans. The simple (in terms of subunit composition) bacterial RNAP is an excellent model system to study the control of gene transcription. This chapter also describes the components of such a system and how they interact to allow regulation of RNAP activity at the level of the DNA opening event (i.e., open complex formation) necessary for trancription initiation.

Communication between Eσ54, promoter DNA and the conserved threonine residue in the GAFTGA motif of the PspF σ54-dependent activator during transcription activation

Conversion of Eσ54 closed promoter complexes to open promoter complexes requires specialized activators which are members of the AAA (ATPases Associated with various cellular Activities) protein family. The ATP binding and hydrolysis activity of Eσ54 activators is used in an energy coupling reaction to remodel the Eσ54 closed promoter complex and to overcome the σ54‐imposed block on open complex formation. The remodelling target for the AAA activator within the Eσ54 closed complex includes a complex interface contributed to by Region I of σ54, core RNA polymerase and a promoter DNA fork junction structure, comprising the Eσ54 regulatory centre. One σ54 binding surface on Eσ54 activators is a conserved sequence known as the GAFTGA motif. Here, we present a detailed characterization of the interaction between Region I of σ54 and the Escherichia coli AAA σ54 activator Phage shock protein F. Using Eσ54 promoter complexes that mimic different conformations adopted by the DNA during open complex formation, we investigated the contribution of the conserved threonine residue in the GAFTGA motif to transcription activation. Our results suggest that the organization of the Eσ54 regulatory centre, and in particular the conformation adopted by the σ54 Region I and the DNA fork junction structure during open complex formation, is communicated to the AAA activator via the conserved T residue of the GAFTGA motif.

Sigma54-dependent transcription activator phage shock protein F of Escherichia coli: a fragmentation approach to identify sequences that contribute to self-association.

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 sigma54-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 sigma54-RNAP (RNA polymerase) closed complex. The self-association determinants within the AAA domain of sigma54-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 sigma54-RNAP closed complex, we suggest that PspF may be organized to link nucleotide interactions and self-association to sigma54-RNAP binding and transcription activation.