Mechanochemical ATPases and transcriptional activation

Abstract

Summary 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.

Investigating protein–protein interfaces in bacterial transcription complexes: a fragmentation approach

“…1). (4,9,10) prokaryotes and archaea and are emerging as an essential class of chaperone-like enzymes. (11,12) Recently, s 54 transcriptional activators, or EBPs, were identified as members of this AAAþ superfamily.…”

“…(18) Bacterial EBPs contain motifs common to AAAþ proteins, and can be divided into seven regions of sequence conservation termed C1 to C7. (9,11) Within region C3, there are two highly conserved sequences of particular importance, the GAFTGA and D/ESELFGH motifs. (9) The D/ESELFGH motif of the Sinorhizobium meliloti C4-dicarboxylic acid transport protein D (DctD) has been demonstrated to be important for crosslinking DctD with s 54 -RNAP in an NTP-independent manner.…”

“…(9,11) Within region C3, there are two highly conserved sequences of particular importance, the GAFTGA and D/ESELFGH motifs. (9) The D/ESELFGH motif of the Sinorhizobium meliloti C4-dicarboxylic acid transport protein D (DctD) has been demonstrated to be important for crosslinking DctD with s 54 -RNAP in an NTP-independent manner. (19,20) It is thought that the integrity of the D/ESELFGH motif may be important for a functional docking site for s 54 , needed for an association between s 54 -RNAP and activator prior to NTP binding or hydrolysis.…”

“…(19,20) It is thought that the integrity of the D/ESELFGH motif may be important for a functional docking site for s 54 , needed for an association between s 54 -RNAP and activator prior to NTP binding or hydrolysis. (9) The C3 region GAFTGA motif has been strongly implicated in biochemical studies in the engagement of s 54 -RNAP during nucleotide hydrolysis, and thus is involved in the coupling of energy, derived from ATP hydrolysis, to the remodelling of s 54 within closed complexes. (9) Identification of a s 54 binding site by protein fragmentation Important questions that arise when considering the functioning of these molecular machines include how NTP binding and hydrolysis lead to different functional states of the AAAþ protein and where the substrate (i.e.…”

“…(9) The C3 region GAFTGA motif has been strongly implicated in biochemical studies in the engagement of s 54 -RNAP during nucleotide hydrolysis, and thus is involved in the coupling of energy, derived from ATP hydrolysis, to the remodelling of s 54 within closed complexes. (9) Identification of a s 54 binding site by protein fragmentation Important questions that arise when considering the functioning of these molecular machines include how NTP binding and hydrolysis lead to different functional states of the AAAþ protein and where the substrate (i.e. s 54 within closed complexes) binding interface lies.…”