A conserved region in the σ⁵⁴‐dependent activator DctD is involved in both binding to RNA polymerase and coupling ATP hydrolysis to activation

Abstract

SummaryRhizobium melioti DctD activates transcription from the dctA promoter by catalysing the isomerization of closed complexes between 54 -RNA polymerase holoenzyme and the promoter to open complexes. DctD must make productive contact with 54 -holoenzyme and hydrolyse ATP to catalyse this isomerization. To define further the activation process, we sought to isolate mutants of DctD that had reduced affinities for 54 -holoenzyme. Mutagenesis was confined to the well-conserved C3 region of the protein, which is required for coupling ATP hydrolysis to open complex formation in 54 -dependent activators. Mutant forms of DctD that failed to activate transcription and had substitutions in the C-terminal half of the C3 region were efficiently cross-linked to 54 and the ␤-subunit of RNA polymerase, suggesting that they bound normally to 54 -holoenzyme. In contrast, some mutant forms of DctD with amino acid substitutions in the N-terminal half of the C3 region had reduced affinities for 54 and the ␤-subunit in the cross-linking assay. These data suggest that the N-terminal half of the C3 region of DctD contains a site that may contact 54-holoenzyme during open complex formation.

Purification and Characterization of the AAA+ Domain ofSinorhizobium melilotiDctD, a σ54-Dependent Transcriptional Activator

“…This pattern of transcripts was not seen for DctD (Fig. 6A), nor was it observed previously for DctD (⌬1-142) (17,43,44). Several lines of evidence argue against these shorter transcripts resulting from contaminating RNase activity.…”

“…Column fractions containing the histidine-tagged proteins were pooled, dialyzed against buffer B (20 mM Tris-HCl [pH 8.8], 20 mM potassium thiocyanate, 5% glycerol, 1 mM dithiothreitol), and applied to a HiTrap Q anion exchange column (5 ml; Pharmacia). The histidine-tagged DctD AAAϩ domain proteins were eluted from the anion exchange column with ϳ200 mM KCl in a linear gradient to 1 M KCl in buffer B. DctD (⌬1-142) and an N-terminal histidine-tagged version of DctD (⌬1-142) were purified as described previously (17,44).…”

“…Removal of the N-terminal regulatory domain of DctD was shown previously to result in a constitutively active form of the protein (6,17). A large collection of mutant forms of DctD that fail to activate transcription has been generated and characterized (5,43,44), and further structural information about these proteins will assist in understanding the function of DctD and other 54 -dependent activators.…”

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

“…(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. (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.…”

Structural studies of conformational changes of proteins upon phosphorylation: Structures of activated CheY, CheY-N16-FliM complex, and AAA ⁺ ATPase domain of NtrC1 in both inactive and active states

“…5.5b). The GAFTGA portion of the insertion into H8 is highly conserved among s 54 activators and has been shown to be involved in coupling the energy available from ATP hydrolysis to a conformational change of s 54 -holoenzyme 34,[37][38][39] . Most mutations that abolish transcriptional activation but not ATP binding or hydrolysis are localized in the GAFTGA sequence (Fig.…”