Hierarchical mechanism of amino acid sensing by the T-box riboswitch

• Published in: Nature communications Vol. 9; no. 1; pp. 1896 - 14
• Main Authors: Suddala, Krishna C., Cabello-Villegas, Javier, Michnicka, Malgorzata, Marshall, Collin, Nikonowicz, Edward P., Walter, Nils G.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 14.05.2018; Nature Publishing Group; Nature Portfolio
• Subjects: 631/57/2265; 631/92/500; Amino acids; Aminoacylation; Anchoring; Base Pairing; Binding; Biosynthesis; Chemoreception; Downstream effects; Fluorescence; Fluorescence microscopy; Gene expression; Gene Expression Regulation, Bacterial; Gram-positive bacteria; Gram-Positive Bacteria - genetics; Gram-Positive Bacteria - metabolism; Humanities and Social Sciences; Microscopy, Fluorescence; Molecular chains; multidisciplinary; Nucleic Acid Conformation; Protein Biosynthesis; Proteins; Riboswitch; Riboswitches; RNA, Bacterial - chemistry; RNA, Bacterial - genetics; RNA, Bacterial - metabolism; RNA, Transfer - chemistry; RNA, Transfer - genetics; RNA, Transfer - metabolism; Science; Science (multidisciplinary); Single Molecule Imaging; Transcription; Transfer RNA Aminoacylation; tRNA
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-018-04305-6

In Gram-positive bacteria, T-box riboswitches control gene expression to maintain the cellular pools of aminoacylated tRNAs essential for protein biosynthesis. Co-transcriptional binding of an uncharged tRNA to the riboswitch stabilizes an antiterminator, allowing transcription read-through, whereas an aminoacylated tRNA does not. Recent structural studies have resolved two contact points between tRNA and Stem-I in the 5′ half of the T-box riboswitch, but little is known about the mechanism empowering transcriptional control by a small, distal aminoacyl modification. Using single-molecule fluorescence microscopy, we have probed the kinetic and structural underpinnings of tRNA binding to a glycyl T-box riboswitch. We observe a two-step mechanism where fast, dynamic recruitment of tRNA by Stem-I is followed by ultra-stable anchoring by the downstream antiterminator, but only without aminoacylation. Our results support a hierarchical sensing mechanism wherein dynamic global binding of the tRNA body is followed by localized readout of its aminoacylation status by snap-lock-based trapping. Riboswitches on 5′ ends of mRNAs are important for bacterial gene regulation. Here the authors probe the mechanism of a tRNA aminoacylation sensing T-box riboswitch using single-molecule fluorescence microscopy to characterize dynamic solution conformations and heterogeneous tRNA binding kinetics.