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.
Highlights
In Gram-positive bacteria, T-box riboswitches control gene expression to maintain the cellular pools of aminoacylated transfer RNAs (tRNAs) essential for protein biosynthesis
Gram-positive bacteria, including pathogenic Bacillus strains, often control the expression of genes involved in maintaining aminoacyl-tRNA pools—aminoacyl-tRNA synthetases, amino acid biosynthesis enzymes, and membrane transporters—through a structure located in the 5′-UTR, or leader, of messenger RNAs (mRNAs) termed a T-box riboswitch[3,4,5]
We show that kfoafsft, kmoffedium, and ksolfofw correspond to tRNA primarily engaging with the Specifier and double T-loop motif (DTM) motifs, the Specifier and antiterminator motifs, and all three motifs, respectively
Summary
In Gram-positive bacteria, T-box riboswitches control gene expression to maintain the cellular pools of aminoacylated tRNAs essential for protein biosynthesis. 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. Gram-positive bacteria, including pathogenic Bacillus strains, often control the expression of genes involved in maintaining aminoacyl-tRNA pools—aminoacyl-tRNA synthetases, amino acid biosynthesis enzymes, and membrane transporters—through a structure located in the 5′-UTR, or leader, of mRNA termed a T-box riboswitch[3,4,5]. Binding of a non-aminoacylated (uncharged) transfer RNA (tRNA) to the T-box in trans is thought to stabilize an antiterminator, promoting transcription read-through by preventing formation of the competing terminator structure[3,4,5] (Fig. 1a). The mechanism of how aminoacylation of tRNA prevents transcription read-through (i.e., antitermination) is unclear
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