Two component signaling systems are very common in bacteria, but also occur in eukaryotes. The signaling occurs through transfer of a phosphate from a kinase to a """"""""receiver"""""""" protein, initiating a conformational change, leading to altered interactions with other proteins to propagate the signal. The investigators will study the bacterial transcription factor NtrC, which upon phosphorylation undergoes self-association, interaction with sigma54 polymerase and ATP hydrolysis, leading to transcription. Understanding this relatively simple system will provide a basis for understanding more complex eukaryotic signaling processes, and could also lead to approaches for interrupting specific bacterial signaling processes. The goal of the work proposed here is to determine the structures of the bacterial transcription factor NtrC, and domains of it, to gain an understanding of the molecular basis for its activity, and thereby the general processes for molecular signal transduction.
Specific aims are to: (1) determine a high resolution structure of the BeF3- activated receiver domain, and to understand differences and similarities in changes upon activation with respect to other receiver domains; (2) to express and purify the wild type sequence DNA binding domain, and determine the structure of this dimer bound to an enhancer DNA sequence, as well as trying to understand the role of the linker connecting to the adjacent domain; (3) to make and screen constructs of the central ATPase domain for structure determination (including those from a thermophile), and determine the structures of this domain in different nucleotide ligation states; (4) to examine the role of the communication helix linking the receiver and central domains, and to analyze broadly the interactions between the receiver and central domains; (5) to examine intact NtrC with gel shift, fluorescence and EPR approaches to track changes upon activation, assembly and nucleotide hydrolysis, and to express the sigma54 subunit of polymerase to start to explore its interactions with NtrC.
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