NIH Research Festival
T-box riboswitches are bacterial cis-regulatory noncoding RNAs that sense and regulate amino acid availability through a multi-partite mRNA-tRNA interaction. T-box RNA is comprised of two conserved domains, a Stem I domain and a 3’ antiterminator domain connected by a flexible linker. Stem I domain recognizes the overall shape and anticodon of a cognate tRNA with sequence and structural specificity. The antiterminator domain detects the molecular volume of the tRNA 3’ end to sense aminoacylation. This readout dictates the formation of either an intrinsic transcription terminator or antiterminator. However, the architecture of a full-length T-box in complex with cognate tRNA and the detailed interactions between the tRNA 3’ region and T-box 3’ domain remain poorly defined. In our study, we develop a novel method to produce, assemble, and isolate stoichiometric complexes of the full-length T-box RNA bound by its cognate tRNA. Using this method, we define a minimal region of the T-box that is necessary and sufficient to selectively bind an uncharged tRNA, and solve the first co-crystal structure of this tRNA-mRNA complex at 2.7 Å by de novo phasing. The structure reveals how tRNA 3’ end is buried inside the antiterminator where a conserved G•U wobble pair at the base of helix A2 abuts the ribose 3’-OH of the tRNA terminal adenosine. This juxtaposition creates steric clash between the universal amino group of the esterified amino acid and the uridine nucleobase, thus providing a general mechanism to reject any aminoacyl-tRNA. We also obtain a cryo-EM structure of the full-length T-box riboswitch in complex with its cognate tRNA at 4.7 Å, an unprecedented resolution for RNA of this size. The EM structure reveals that T-box Stem I and the newly defined 3’ domains simultaneously bind two faces of the cognate tRNA, orchestrated and facilitated by a surprisingly ordered inter-domain single-stranded linker. Taken together, the structures show that intermolecular stacking allows Stem I and antiterminator domains of the T-box mRNA to sandwich the uncharged tRNA to stabilize the antiterminator to transcribe downstream genes. These novel structures provide a detailed mechanistic framework of how a RNA-based steric device can achieve selectivity for a cognate, uncharged tRNA. This work informs the development of new avenues and targets to battle the global epidemic of antibiotic resistance, and strategies to modulate human microbiota to improve health.
Scientific Focus Area: Structural Biology
This page was last updated on Friday, March 26, 2021