Two transcriptionally active OmpR mutants that do not require phosphorylation by EnvZ in an Escherichia coli cell-free system

Abstract

D55Q-T83A and D55Q-G94S, two pseudorevertants of the D55Q mutant OmpR, an Escherichia coli transcriptional activator, were isolated previously by R. Brissette, K. Tsung, and M. Inouye (J. Bacteriol. 173:3749-3755, 1991). These pseudorevertant OmpR proteins were purified and examined for their function as transcriptional activators in a cell-free system with an ompF DNA fragment. These proteins were transcriptionally active, even after acid treatment, whereas the wild-type OmpR was completely inactive after the same treatment. Phosphorylation of acid-treated wild-type OmpR with an EnvZ11 membrane fraction and ATP restored transcriptional activity, whereas the activities of the mutant OmpR proteins did not change after phosphorylation.

Structural and functional analyses of activating amino acid substitutions in the receiver domain of NtrC: Evidence for an activating surface

“…see Olmedo et al, 1990;Speigelman et al, 1990) or low molecular mass phosphodonors such as acetyl or carbamoyl phosphate (Feng et al, 1992). Although similar selections have been employed to isolate mutant forms of many other proteins containing receiver domains (Bourret et al, 1990;Bowrin et al, 1992;Flashner et al, 1995;Hirschman et al, 1985;Ireton et al, 1993;Jin et al, 1993;Kahn & Ditta, 1991;Pazour et al, 1992;Shoji et al, 1988;Speigelman et al, 1990;Stewart, 1993;Stewart et al, 1990), relatively few have been studied in vitro to identify those that mimic activation in the absence of phosphorylation (Bowrin et al, 1992;Da Re et al, 1994;Gu et al, 1994;Han & Winans, 1994;Klose et al, 1993;Moore et al, 1993;Stewart, 1993;Welch et al, 1994;Zhu et al, 1996).…”

Involvement of the amino‐terminal phosphorylation module of UhpA in activation of uhpT transcription in Escherichia coli

“…However, the surface of CheY that interacts with CheA lies on the opposite side of the module from helix-1, between sheet 4 and helix-5 (Shukla and Matsumura, 1995). This surface overlaps the residues that interact with the FliM switch (Roman et al, 1992), and includes residues at which changes result in phosphorylation independence in OmpR (Bowrin et al, 1992) and NtrC (Flashner et al, 1995). On the other hand, substitutions in helix-1 of CheY at the positions corresponding to Q19 and G22 of UhpA result in a reduced response to the CheZ co-phosphatase (Sanna et al, 1995).…”

The α subunit of RNA polymerase specifically inhibits expression of the porin genes ompF and ompC in vivo and in vitro in Escherichia coli

“…[25] was transcribed in a cell-free system in the presence of OmpR and a or o-. Bowrin et al [27]. Bowrin et al [27].…”

“…A 330-bp XbaI-BamHI fragment from plasmid pKI0033 [25] and a 322-bp BamHI-HindIII fragment from pKEN022 [26] were used as templates in a cell-free transcription system as described by Bowrin et al [27] with slight modifications. Puri- RNA transcripts were analyzed on a 8%/8 M urea/polyacrylamide gel.…”

“…The effects of the a subunit on ompF transcription were further examined in a cell-free system using a 330-bp XbaI-BamHI promoter fragment from pKI0033 [25] as previously described by Bowrin et al [27] with slight modifications. Purified a/~r (50-100 rig) were added to the template prior to the addition of OmpR.…”