Nucleotide-Dependent Conformational Changes in the σ⁵⁴-Dependent Activator DctD

Abstract

Activators of54 -RNA polymerase holoenzyme couple ATP hydrolysis to formation of an open promoter complex. DctD ⌬1-142 , a truncated and constitutively active form of the 54 -dependent activator DctD from Sinorhizobium meliloti, displayed an altered DNase I footprint at its binding site located upstream of the dctA promoter in the presence of ATP. The altered footprint was not observed for a mutant protein with a substitution at or near the putative arginine finger, a conserved arginine residue thought to contact the nucleotide. These data suggest that structural changes in DctD ⌬1-142 during ATP hydrolysis can be detected by alterations in the DNase I footprint of the protein and may be communicated by interactions between bound nucleotide and the arginine finger. In addition, kinetic data for changes in fluorescence energy transfer upon binding of 2(3)-O-(N-methylanthraniloyl)-ATP (Mant-ATP) to DctD ⌬1-142 and DctD suggested that these proteins undergo multiple conformational changes following ATP binding.

The crystal structure of apo -FtsH reveals domain movements necessary for substrate unfolding and translocation

“…2B). This change concurs with earlier studies (7,34). This more open conformation of the AAA domain is incompatible with the C2-symmetric packing observed in the ADP-bound state.…”

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

“…These data suggest a role for the DNA-binding domain in preventing assembly of DctD into a functional oligomeric complex in the absence of binding to the UAS, implying that the DNA-binding domain interacts with the AAAϩ domain. DNase I footprinting of DctD (⌬1-142) at the dctA UAS revealed changes in the footprint in response to ADP ⅐ AlF x , consistent with the idea that the DNA-binding domain can communicate with the AAAϩ domain, at least in the transition state during ATP hydrolysis (45). Alternatively, intermolecular interactions between the DNA-binding domains of DctD monomers may interfere with assembly of a functional oligomeric complex, which may be supported by the observation that introduction of three alanine substitutions in the enhancer recognition helix of S. enterica serovar Typhimurium NtrC eliminates DNAbinding activity and stabilizes the dimeric state of the protein (26).…”

The structural basis for regulated assembly and function of the transcriptional activator NtrC

“…At high concentrations many of the 54 -dependent ATPase domains (including NtrC's) can act in the absence of DNAbinding activity and in an enhancer-independent fashion (North and Kustu 1997). However, substitutions in the GAFTGA loop of NtrC caused binding to the enhancer or even nonspecific DNA to act as a negative allosteric regulator of the interaction with RNA polymerase , and prior studies of DctD, activated by deleting its N-terminal receiver domain, showed that ADP-AlF x , but not ADP or ADP-BeF x , caused hypersensitivity to DNase I to appear between the tandem binding sites in the dctA UAS (Wang et al 2003). These effects may be related to the change in relative flexibility in the connection of the DNA-binding domains with the ATPase ring that we observed during the hydrolysis cycle, which would have a dramatic effect on the structure of the DNA.…”

“…ADP-AlF x , frequently used as a transition state analog for ATP hydrolysis, is known to stabilize the ring form of PspF and its complex with 54 (Chaney et al 2001), and to alter the protein/DNA footprint of an activated form of DctD (Wang et al 2003). Therefore a new full data set was obtained for activated NtrC (S160F, 3-Ala) in the presence of ADP-AlF x .…”