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J. Biol. Chem., Vol. 281, Issue 42, 31832-31842, October 20, 2006

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Comprehensive Alanine-scanning Mutagenesis of Escherichia coli CsrA Defines Two Subdomains of Critical Functional Importance^*

Jeffrey Mercante, Kazushi Suzuki, Xiaodong Cheng, Paul Babitzke^¶, and Tony Romeo¹

From the Departments of Microbiology and Immunology and Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322 and the ^¶Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802

The RNA-binding protein CsrA (carbon storage regulator) of Escherichia coli is a global regulator of gene expression and is representative of the CsrA/RsmA family of bacterial proteins. These proteins act by regulating mRNA translation and stability and are antagonized by binding to small noncoding RNAs. Although the RNA target sequence and structure for CsrA binding have been well defined, little information exists concerning the protein requirements for RNA recognition. The three-dimensional structures of three CsrA/RsmA proteins were recently solved, revealing a novel protein fold consisting of two interdigitated monomers. Here, we performed comprehensive alanine-scanning mutagenesis on csrA of E. coli and tested the 58 resulting mutants for regulation of glycogen accumulation, motility, and biofilm formation. Quantitative effects of these mutations on expression of glgCA`-'lacZ, flhDC `-'lacZ, and pgaA`-'lacZ translational fusions were also examined, and eight of the mutant proteins were purified and tested for RNA binding. These studies identified two regions of the amino acid sequence that were critical for regulation and RNA binding, located within the first (₁, residues 2-7) and containing the last (₅, residues 40-47) -strands of CsrA. The ₁ and ₅ strands of opposite monomers lie adjacent and parallel to each other in the three-dimensional structure of this protein. Given the symmetry of the CsrA dimer, these findings imply that two distinct RNA binding surfaces or functional subdomains lie on opposite sides of the protein.

Received for publication, June 23, 2006 , and in revised form, August 15, 2006.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1s and 2s.

¹ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Emory University School of Medicine, 3105 Rollins Research Center, 1510 Clifton Rd. N.E., Atlanta, GA 30322. Tel.: 404-727-3734; Fax: 404-727-3659; E-mail: romeo{at}microbio.emory.edu.

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