Constitutive ATP hydrolysis and transcription activation by a stable, truncated form of Rhizobium meliloti DCTD, a σ⁵⁴-dependent transcriptional activator

The dctD gene product (DCTD) activates transcription from dctA by the σ⁵⁴-holoenzyme form of RNA polymerase in Rhizobium meliloti. We have purified a constitutively active form of R. meliloti DCTD that lacks 142 amino acid residues from the N terminus (designated DCTD_L143). Purified DCTD_L143 recognized the DCTD-binding sites at the dctA promoter region and catalyzed the isomerization of closed complexes between σ⁵⁴-holoenzyme and the dctA promoter to open complexes. Like the related σ⁵⁴-dependent activators NTRC and NIFA, a purine nucleoside triphosphate with a hydrolyzable β-γ bond was required prior to transcription initiation for this isomerization. DCTD_L143 hydrolyzed purine nucleoside triphosphates but not pyrimidine nucleoside triphosphates. As observed with NTRC-phosphate, the specific activity for the ATPase of DCTD_L143 was strongly dependent on the enzyme concentration and was stimulated by DNA fragments bearing the binding sites for the protein. These DNA fragments increased the V_max for MgATP hydrolysis but did not significantly lower the apparent K_m for MgATP. These data are consistent with the idea proposed for related activators that DCTD_L143 must assemble into an active, oligomeric form before it can hydrolyze MgATP and presumably activate transcription.