Sinorhizobium meliloti DctD is an activator of σ54-RNA polymerase holoenzyme and member of the AAA+ superfamily of ATPases. DctD uses energy released from ATP hydrolysis to stimulate the isomerization of a closed promoter complex to an open complex. DctD binds to upstream activation sequences (UAS) and contacts the closed complex through DNA looping to activate transcription, but the UAS is not essential for activation if DctD is expressed at higher than normal levels. Introduction of specific substitutions within or near the conserved ESELFG motif in the C3 region of a truncated, constitutively active form of DctD produced several mutant forms of the protein that had increased dependence on the UAS for activation. Removing the DNA-binding domain from one UAS-dependent mutant and from one activation-deficient mutant significantly increased transcriptional activation, indicating that the DNA-binding domain interfered with the activities of these mutant proteins. A UAS-dependent mutant with a P315L substitution in the CS region was identified from a genetic screen. Alanine scanning mutagenesis of conserved amino acid residues around Pro-315 produced two additional UAS-dependent mutants as well as several mutants that failed to activate transcription but retained ATPase activity. In contrast to the two mutant proteins with substitutions in the C3 region, removal of the DNA-binding domain from the mutant proteins with substitutions in the C6 region did not stimulate their activity. The residues in the CS region that were altered are in a probable hinge region between the α/β and α-helical subdomains of the AAA+ domain. The α-helical subdomain contains the sensor II helix that has been implicated in other AAA+ proteins as sensing changes in the nucleotide during the hydrolysis cycle. Substitutions in the hinge region may have abolished nucleotide sensing by interfering with sub-domain interactions, altering the relative orientation of the sensor II helix or interfering with oligomerization of the protein.
All Science Journal Classification (ASJC) codes
- Molecular Biology