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.

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. Structure studies of the 100 nt stem I domain reveal tRNA utilizes base pairing and stacking interactions to bind the stem, but little is known structurally about the 180 nt riboswitch core (stem I, stem III, and antiterminator stem) in complex with tRNA and 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 B. subtilis and O. iheyensis glycyl T-box and T-box:tRNA constructs. The data reveal that tRNA:riboswitch core binding at equilibrium involves only Specifier-anticodon and anti-terminator-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 anti-terminator 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.

An RNA oligonucleotide that contains the binding site for Escherichia coli ribosomal protein S8 was prepared with uniform 15N isotopic enrichment and uniform deuterium enrichment at all non-exchangeable sites using enzymatic methods. The RNA binding site, which contains 44 nt, forms a hairpin in solution and requires Mg2+for proper folding. The longitudinal magnetization recovery rates of the exchangeable protons were compared for the [2H,15N]-enriched RNA molecule and for the corresponding fully [2H,15N]-enriched RNA hairpin. It was found that 1H-1H dipolar relaxation significantly contributes to the recovery of exchangeable proton longitudinal magnetization. The exchangeable proton resonance line widths were less affected by deuteration, indicating that chemical exchange with H2O remains the dominant mechanism of transverse magnetization relaxation. Nevertheless, deuteration of this RNA hairpin was found to enhance the sensitivity of NOE-based experiments relative to the fully protonated hairpin and to simplify 2D NMR spectra. The increased signal-to-noise ratio facilitated the assignment of the cytidine amino resonances and several of the purine nucleotide amino resonances and permitted the identification of NOE crosspeaks that could not be observed in spectra of the fully protonated RNA hairpin.

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The bacterial S8 protein is a ribosomal protein which binds to the 16S rRNA in the central domain and plays a critical role in 30S ribosomal subunit assembly. An RNA aptamer with high affinity for B. anthracis S8 protein was developed using the SELEX methodology and crystals of the complex were obtained. To understand how S8/RNA recognition occurs for this apatamer, we are determining the structure of the complex by X‐ray crystallography. Diffraction data for the S8/RNA crystal were collected to 2.6Å resolution at the Advanced Light Source synchrotron (Berkeley, CA). The crystal belonged to the space group P212121 with unit cell parameters a= 55.41 b=59.27 c=92.25Å, α=90, β= 90, γ=90. The data were processed using the program d*TREK with Rmerge=9.2 % and completeness = 99.9 %. A molecular replacement solution for S8/RNA complex was found with the program Phaser for MR from the CCP4 suite using S8 protein (unpub.data) as a search model. The initial solution suggested one monomer per asymmetric unit consistent with a Matthews coefficient of 3.2 (65% solvent). The molecular replacement solution was further confirmed by the initial (2Fo‐Fc) maps generated using Coot that clearly indicated electron density for the RNA aptamer. A model is being constructed for the protein‐RNA complex. This work was supported by NSF (0641792) to Y.S. and The Robert A. Welch Foundation (C‐1584 to Y.S. and C‐1277 to E.P.N.) and NIH(U54 AI057156).