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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM062163-01
Application #
6225792
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Lewis, Catherine D
Project Start
2001-01-01
Project End
2004-12-31
Budget Start
2001-01-01
Budget End
2001-12-31
Support Year
1
Fiscal Year
2001
Total Cost
$216,260
Indirect Cost
Name
Lawrence Berkeley National Laboratory
Department
Biochemistry
Type
Organized Research Units
DUNS #
078576738
City
Berkeley
State
CA
Country
United States
Zip Code
94720
Siegel, Alexander R; Wemmer, David E (2016) Role of the ?54 Activator Interacting Domain in Bacterial Transcription Initiation. J Mol Biol 428:4669-4685
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Batchelor, Joseph D; Lee, Peter S; Wang, Andrew C et al. (2013) Structural mechanism of GAF-regulated ýý(54) activators from Aquifex aeolicus. J Mol Biol 425:156-70
Vidangos, Natasha; Maris, Ann E; Young, Anisa et al. (2013) Structure, function, and tethering of DNA-binding domains in ??? transcriptional activators. Biopolymers 99:1082-96
Sterling, Harry J; Batchelor, Joseph D; Wemmer, David E et al. (2010) Effects of buffer loading for electrospray ionization mass spectrometry of a noncovalent protein complex that requires high concentrations of essential salts. J Am Soc Mass Spectrom 21:1045-9
Batchelor, Joseph D; Sterling, Harry J; Hong, Eunmi et al. (2009) Receiver domains control the active-state stoichiometry of Aquifex aeolicus sigma54 activator NtrC4, as revealed by electrospray ionization mass spectrometry. J Mol Biol 393:634-43
Hong, Eunmi; Doucleff, Michaeleen; Wemmer, David E (2009) Structure of the RNA polymerase core-binding domain of sigma(54) reveals a likely conformational fracture point. J Mol Biol 390:70-82
Batchelor, Joseph D; Doucleff, Michaeleen; Lee, Chul-Jin et al. (2008) Structure and regulatory mechanism of Aquifex aeolicus NtrC4: variability and evolution in bacterial transcriptional regulation. J Mol Biol 384:1058-75
Doucleff, Michaeleen; Pelton, Jeffrey G; Lee, Peter S et al. (2007) Structural basis of DNA recognition by the alternative sigma-factor, sigma54. J Mol Biol 369:1070-8
Fraser, James S; Merlie Jr, John P; Echols, Nathaniel et al. (2007) An atypical receiver domain controls the dynamic polar localization of the Myxococcus xanthus social motility protein FrzS. Mol Microbiol 65:319-32

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