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Updated: Aug 29, 2026

DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems
Published on: July 21, 2014
A search for amino acid substitutions that universally activate response regulators
Jenny G Smith1, Jamie A Latiolais, Gerald P Guanga
1Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599-7290, USA.
Abstract:
Two-component regulatory systems, typically composed of a sensor kinase to detect a stimulus and a response regulator to execute a response, are widely used by microorganisms for signal transduction. Response regulators exhibit a high degree of structural similarity and undergo analogous activating conformational changes upon phosphorylation. The activity of particular response regulators can be increased by specific amino acid substitutions, which either prolong the lifetime or mimic key features of the phosphorylated state. We probed the universality of response regulator activation by amino acid substitution. Thirty-six mutations that activate 11 different response regulators were identified from the literature. To determine whether the activated phenotypes would be retained in the context of a different response regulator, we recreated 51 analogous amino acid substitutions at corresponding positions of CheY. About 55% of the tested substitutions completely or partially inactivated CheY, approximately 30% were phenotypically silent, and approximately 15% activated CheY. Three previously uncharacterized activated CheY mutants were found. The 94NS (and presumably 94NT) substitutions resulted in resistance to CheZ-mediated dephosphorylation. The 113AP substitution led to enhanced autophosphorylation and may increase the fraction of non-phosphorylated CheY molecules that populate the activated conformation. The locations of activating substitutions on the response regulator three-dimensional structure are generally consistent with current understanding of the activation mechanism. The best candidates for potentially universal activating substitutions of response regulators identified in this study were 13DK and 113AP.
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