tRNA regulation of gene expression: Interactions of an mRNA 5 '-UTR with a regulatory tRNA

摘要

Many genes encoding aminoacyl-tRNA synthetases and other amino acid-related products in Gram-positive bacteria, including important pathogens, are regulated through interaction of unacylated tRNA with the 5'-untranslated region (5'-UTR) of the mRNA. Each gene regulated by this mechanism responds specifically to the cognate tRNA, and specificity is determined by pairing of the anticodon of the tRNA with a codon sequence in the "Specifier Loop'' of the 5'-UTR. For the 5'-UTR to function in gene regulation, the mRNA folding interactions must be sufficiently stable to present the codon sequence for productive binding to the anticodon of the matching tRNA. A model bimolecular system was developed in which the interaction between two half molecules ("Common'' and "Specifier'') would reconstitute the Specifier Loop region of the 5'-UTR of the Bacillus subtilis glyQS gene, encoding GlyRS mRNA. Gel mobility shift analysis and fluorescence spectroscopy yielded experimental K(d)s of 27.6 +/- 1.0 mu M and 10.5 +/- 6 0.7 mu M, respectively, for complex formation between Common and Specifier half molecules. The reconstituted 5'-UTR of the glyQS mRNA bound the anticodon stem and loop of tRNA Gly (ASL Gly GCC) specifically and with a significant affinity (K-d = 20.2 +/- 6 1.4 mu M). Thus, the bimolecular 5'-UTR and ASL(GCC)(Gly) models mimic the RNA-RNA interaction required for T box gene regulation in vivo.