T box bacterial genes utilize a riboswitch mechanism to regulate gene expression at the transcriptional level. Complementary base pairing of the 5'-untranslated mRNA with uncharged cognate tRNA stabilizes formation of an antiterminator element and permits complete transcription. In the absence of tRNA, a mutually exclusive RNA terminator element forms and results in transcription termination. This regulatory mechanism requires divalent metal ions at the antitermination event. The structural effects of Mg(2+) binding to antiterminator model RNA were investigated to ascertain if this requirement is due to the presence of a specific metal ion binding site in the antiterminator. Spectroscopic analysis identified the presence of a hydrated, diffuse Mg(2+) binding site. The results indicate that the mechanistic requirement for divalent metal ions is not due to Mg(2+)-induced pre-formation of a functional antiterminator receptor; rather, Mg(2+) binds in a helical region of high phylogenetic sequence conservation adjacent to the tRNA binding site.