title: Direct evaluation of tRNA aminoacylation status by the T-box riboswitch using tRNA-mRNA stacking and steric readout.
abstract: T-boxes are gene-regulatory mRNA elements with which Gram-positive bacteria sense amino acid availability. T-boxes have two functional domains. Stem I recognizes the overall shape and anticodon of tRNA, while a 3' domain evaluates its aminoacylation status, overcoming an otherwise stable transcriptional terminator if the bound tRNA is uncharged. Although T-boxes are believed to evaluate tRNA charge status without using any proteins, this has not been demonstrated experimentally because of the instability of aminoacyl-tRNA. Using a simple method to prepare homogeneous aminoacyl-tRNA, we show that the Bacillus subtilis glyQS T-box functions independently of any tRNA-binding protein. Comparison of aminoacyl-tRNA analogs demonstrates that the T-box detects the molecular volume of tRNA 3'-substituents. Calorimetry and fluorescence lifetime analysis of labeled RNAs shows that the tRNA acceptor end coaxially stacks on a helix in the T-box 3' domain. This intimate intermolecular association, selective for uncharged tRNA, stabilizes the antiterminator conformation of the T-box.
authors: National Heart, Lung and Blood Institute, 50 South Drive, MSC 8012, Bethesda, MD 20892-8012, USA.
authors: Jinwei
authors: J
authors: Zhang
authors: National Heart, Lung and Blood Institute, 50 South Drive, MSC 8012, Bethesda, MD 20892-8012, USA. Electronic address: adrian.ferre@nih.gov.
authors: Adrian R
authors: AR
authors: Ferré-D'Amaré
mesh_terms: D049148:Aminoacylation; D001412:Bacillus subtilis / Q000235:genetics*; D012333:RNA, Messenger; D012343:RNA, Transfer / Q000378:metabolism*; D058928:Riboswitch / Q000502:physiology*
chemical_list: D012333:RNA, Messenger; D058928:Riboswitch; D012343:RNA, Transfer