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Title: The magic spot: identification of the binding site for ppGpp on E. coli RNA polymerase

Author
item ROSS, WILMA - University Of Wisconsin
item Vrentas, Catherine
item SANCHEZ-VAZQUEZ, PATRICIA - University Of Wisconsin
item GAAL, TAMAS - University Of Wisconsin
item GOURSE, RICHARD - University Of Wisconsin

Submitted to: Molecular Cell
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/18/2013
Publication Date: 5/9/2013
Citation: Ross, W., Vrentas, C.E., Sanchez-Vazquez, P., Gaal, T., Gourse, R.L. 2013. The magic spot: a ppGpp binding site on E. coli RNA polymerase responsible for regulation of transcription initiation. Molecular Cell. 50(3):420-429.

Interpretive Summary: As bacteria face environments with different levels of available nutrients, each bacterial cell must adapt its metabolism to the current conditions. For example, when a bacterial cell is starving for amino acids (the building blocks of proteins), it shuts down its metabolism, including the production of the ribosomal components involved in protein production. The molecule ppGpp, or "magic spot", plays a key role in this process, known as the "stringent response." By binding to the protein complex RNA polymerase (as well as other enzymes), ppGpp plays a key role in controlling the production of ribosomal RNA as well as modulating the virulence of many bacterial pathogens. While the effects of ppGpp production in the cell have been well-characterized at a physiological and molecular level, many questions still remain about the location at which ppGpp interacts with the large RNA polymerase complex to cause these effects. Here, we utilize a protein-ppGpp crosslinking approach to identify the location of the ppGpp binding site on Escherichia coli RNA polymerase. Changes (mutations) to this site prevent RNA polymerase from binding to ppGpp and impact the metabolism of the bacterial cell. This work provides the field with new insights about how the interaction of ppGpp with RNA polymerase leads to the molecular changes critical to the regulation of metabolism. The work will also be useful in understanding the response of many different taxa (groups) of bacteria to ppGpp, and can potentially be applied to future investigations designed to modulate bacterial growth and virulence.

Technical Abstract: Despite more than 40 years of study of the global regulatory nucleotide ppGpp ("magic spot") in Escherichia coli, its target site on RNA polymerase (RNAP), and therefore its mechanism of action, is unknown. We report here a binding site for ppGpp on E. coli RNAP, identified by crosslinking, protease mapping, and analysis of mutant RNAPs failing to respond to ppGpp. Strains with mutant ppGpp binding sites display properties characteristic of cells defective for ppGpp synthesis. The binding site is at an interface of two RNAP subunits, omega and beta prime, and its position suggests an allosteric mechanism involving restriction of motion between two mobile modules in RNAP. Identification of the binding site allows prediction of bacterial species that respond to ppGpp by direct targeting of RNAP.