Solution Structure of the K-Turn and Specifier Loop Domains from the Bacillus subtilis tyrS T-Box Leader RNA

Abstract

In Gram-positive bacteria, the RNA transcripts of many amino acid biosynthetic and aminoacyl tRNA synthetase genes contain 5′ untranslated regions, or leader RNAs, that function as riboswitches. These T-box riboswitches bind cognate tRNA molecules and regulate gene expression by a transcription attenuation mechanism. The Specifier Loop domain of the leader RNA contains nucleotides that pair with nucleotides in the tRNA anticodon loop and is flanked on one side by a kink-turn (K-turn), or GA, sequence motif. We have determined the solution NMR structure of the K-turn sequence element within the context of the Specifier Loop domain. The K-turn sequence motif has several noncanonical base pairs typical of K-turn structures but adopts an extended conformation. The Specifier Loop domain contains a loop E structural motif, and the single-strand Specifier nucleotides stack with their Watson–Crick edges displaced toward the minor groove. Mg2+ leads to a significant bending of the helix axis at the base of the Specifier Loop domain, but does not alter the K-turn. Isothermal titration calorimetry indicates that the K-turn sequence causes a small enhancement of the interaction between the tRNA anticodon arm and the Specifier Loop domain. One possibility is that the K-turn structure is formed and stabilized when tRNA binds the T-box riboswitch and interacts with Stem I and the antiterminator helix. This motif in turn anchors the orientation of Stem I relative to the 3′ half of the leader RNA, further stabilizing the tRNA–T box complex.