It is impossible to predict which pathway, direct glutaminylation of tRNA Gln or tRNA-dependent transamidation of glutamyl-tRNA Gln , generates mitochondrial glutaminyl-tRNA Gln for protein synthesis in a given species. The report that yeast mitochondria import both cytosolic glutaminyl-tRNA synthetase and tRNA Gln has challenged the widespread use of the transamidation pathway in organelles. Here we demonstrate that yeast mitochondrial glutaminyl-tRNA Gln is in fact generated by a transamidation pathway involving a novel type of trimeric tRNAdependent amidotransferase (AdT). More surprising is the fact that cytosolic glutamyl-tRNA synthetase ( c ERS) is imported into mitochondria, where it constitutes the mitochondrial nondiscriminating ERS that generates the mitochondrial mischarged glutamyl-tRNA Gln substrate for the AdT. We show that dual localization of c ERS is controlled by binding to Arc1p, a tRNA nuclear export cofactor that behaves as a cytosolic anchoring platform for c ERS. Expression of Arc1p is down-regulated when yeast cells are switched from fermentation to respiratory metabolism, thus allowing increased import of c ERS to satisfy a higher demand of mitochondrial glutaminyltRNA Gln for mitochondrial protein synthesis. This novel strategy that enables a single protein to be localized in both the cytosol and mitochondria provides a new paradigm for regulation of the dynamic subcellular distribution of proteins between membrane-separated compartments.
T-box riboswitches control transcription of downstream genes through the tRNA-binding formation of terminator or antiterminator structures. Previously reported T-boxes were described as single-specificity riboswitches that can bind specific tRNA anticodons through codon–anticodon interactions with the nucleotide triplet of their specifier loop (SL). However, the possibility that T-boxes might exhibit specificity beyond a single tRNA had been overlooked. In Clostridium acetobutylicum , the T-box that regulates the operon for the essential tRNA-dependent transamidation pathway harbors a SL with two potential overlapping codon positions for tRNA Asn and tRNA Glu . To test its specificity, we performed extensive mutagenic, biochemical, and chemical probing analyses. Surprisingly, both tRNAs can efficiently bind the SL in vitro and in vivo. The dual specificity of the T-box is allowed by a single base shift on the SL from one overlapping codon to the next. This feature allows the riboswitch to sense two tRNAs and balance the biosynthesis of two amino acids. Detailed genomic comparisons support our observations and suggest that “flexible” T-box riboswitches are widespread among bacteria, and, moreover, their specificity is dictated by the metabolic interconnection of the pathways under control. Taken together, our results support the notion of a genome-dependent codon ambiguity of the SLs. Furthermore, the existence of two overlapping codons imposes a unique example of tRNA-dependent regulation at the transcriptional level.
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