ABSTRACT
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.
Subject(s)
Gene Expression Regulation, Bacterial , Gram-Positive Bacteria/genetics , RNA, Bacterial/chemistry , RNA, Transfer/chemistry , Riboswitch , Base Pairing , Gram-Positive Bacteria/metabolism , Microscopy, Fluorescence , Nucleic Acid Conformation , Protein Biosynthesis , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolism , Single Molecule Imaging , Transfer RNA AminoacylationABSTRACT
In Gram-positive bacteria, the tRNA-dependent T-box riboswitch system regulates expression of amino acid biosynthetic and aminoacyl-tRNA synthetase genes through a transcription attenuation mechanism. Binding of uncharged tRNA "closes" the switch, allowing transcription read-through. Structural studies of the 100-nucleotide stem I domain reveal tRNA utilizes base pairing and stacking interactions to bind the stem, but little is known structurally about the 180-nucleotide riboswitch core (stem I, stem III, and antiterminator stem) in complex with tRNA or the mechanism of coupling of the intermolecular binding domains crucial to T-box function. Here we utilize solution structural and biophysical methods to characterize the interplay of the different riboswitch-tRNA contact points using Bacillus subtilis and Oceanobacillus iheyensis glycyl T-box and T-box:tRNA constructs. The data reveal that tRNA:riboswitch core binding at equilibrium involves only Specifier-anticodon and antiterminator-acceptor stem pairing. The elbow:platform stacking interaction observed in studies of the T-box stem I domain is released after pairing between the acceptor stem and the bulge in the antiterminator helix. The results are consistent with the model of T-box riboswitch:tRNA function in which tRNA is captured by stem I of the nascent mRNA followed by stabilization of the antiterminator helix and the paused transcription complex.