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United States Department of Agriculture

Agricultural Research Service

Research Project: TRANSMISSION, DIFFERENTIATION, AND PATHOBIOLOGY OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES Title: Direct interactions between the coiled-coil tip of DksA and the trigger loop of RNA polymerase mediate transcriptional regulation

Authors
item Lennon, Christopher -
item Ross, Wilma -
item Martin-Tumasz, Steve -
item Toulokhonov, Innokenti -
item Vrentas, Catherine
item Rutherford, Steven -
item Lee, Jeong-Hyun -
item Butcher, Samuel -
item Gourse, Richard -

Submitted to: Genes and Development
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 10, 2012
Publication Date: December 1, 2012
Citation: Lennon, C.W., Ross, W., Martin-Tumasz, S., Toulokhonov, I., Vrentas, C.E., Rutherford, S.T., Lee, J.H., Butcher, S.E., Gourse, R.L. 2012. Direct interactions between the coiled-coil tip of DksA and the trigger loop of RNA polymerase mediate transcriptional regulation. Genes and Development. 26(23):2634-2646.

Interpretive Summary: Transcription in the bacterial cell is a key intermediate step in the production of proteins (which provide structure and internal functions to the cell) from the code that is carried in the cells' genetic material. Proper regulation of this process by the cell, which includes changes in the amount and types of proteins that eventually accumulate inside the cell, is critical in order for bacteria to adapt to changes in environment. A protein called DksA has previously been shown to be important in the adaptation of bacterial cell transcription to changes in the level of available nutrients. A more detailed understanding of the response of bacteria to changes in nutrient levels is important for future studies in the control of bacterial proliferation and in the understanding of bacterial virulence (ability to cause disease). Here, we examine in molecular detail the interaction of the DksA protein with RNA Polymerase (RNAP), a complex of proteins that is central to carrying out the transcription process. We provide, for the first time, a three dimensional model of how these protein complexes interact inside the Escherichia coli cell to affect the transcription process. The results of this research can be applied to further studies of the adaptation of bacteria to nutrient stress, including the interaction of these proteins with a small signaling molecule called "magic spot" that is produced in the bacterial cell under nutrient starvation conditions.

Technical Abstract: E. coli DksA is in a class of transcription factors that modify RNA polymerase (RNAP) in all three kingdoms of life. DksA potentiates the effects of the global regulator ppGpp and the initiating NTP, controlling transcription initiation without binding to DNA. Incorporating benzoyl-phenylalanine (Bpa) at specific positions in DksA and mapping its crosslinks to RNAP allowed computational docking of the two proteins. The resulting evidenced-based model of the DksA-RNAP complex confirmed that DksA binds to the RNAP secondary channel, defined the orientation of DksA in the channel, and predicted DksA interactions with the rim helices and trigger loop (TL). Protein-protein footprinting, effects of RNAP variants on DksA function, and crosslinks of RNAP-Bpa to DksA supported these predictions. Engineered cysteine substitutions resulted in disulfide bonds between the TL and DksA coiled-coil tip, providing an explanation for the essentiality of specific residues in the DksA tip and TL for regulation by DksA/ppGpp.

Last Modified: 10/30/2014