Alanine Scanning Mutagenesis of Anti-TRAP (AT) Reveals Residues Involved in Binding to TRAP

The trp RNA-binding attenuation protein (TRAP) regulates expression of the tryptophan biosynthetic (trp) genes in response to changes in intracellular levels of free l-tryptophan in many Gram-positive bacteria. When activated by binding tryptophan, TRAP binds to the mRNAs of several genes involved in tryptophan metabolism, and down-regulates transcription or translation of these genes. Anti-TRAP (AT) is an antagonist of TRAP that binds to tryptophan-activated TRAP and prevents it from binding to its RNA targets, and thereby up-regulates trp gene expression. The crystal structure shows that AT is a cone-shaped trimer (AT₃) with the N-terminal residues of the three subunits assembled at the apex of the cone and that these trimers can further assemble into a dodecameric (AT₁₂) structure. Using alanine-scanning mutagenesis we found four residues, all located on the "top" region of AT₃, that are essential for binding to TRAP. Fluorescent labeling experiments further suggest that the top region of AT is in close juxtaposition to TRAP in the AT-TRAP complex. In vivo studies confirmed the importance of these residues on the top of AT in regulating TRAP mediated gene regulation.