Two-codon T-box riboswitch binding two tRNAs

Abstract

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.

Ancient translation factor is essential for tRNA-dependent cysteine biosynthesis in methanogenic archaea

“…The ND-aaRSs and AdTs can form complexes called transamidosomes (28), which could allow channeling of the mischarged aa-tRNAs from the ND-aaRSs to the AdTs without dissociation from the complexes (29,30). Several different types of transamidosome and regulatory features have been discovered (31). (i) The Asn transamidosome in Thermus thermophilus requires tRNA Asn as a scaffold for formation and is stable over the sequential reactions (28,29).…”

Induced Transcriptional Expression of <i>Bacillus subtilis</i> Amino Acid Permease <i>yvbW</i> in Response to Leucine Limitation

“…A CUC leucine codon is located directly downstream of the GAA, placing the specifier at the consensus position, a single base from the lower helix. The work of Saad et al [10] demonstrated that the specifier sequence can shift allowing more than one codon to serve as the specifier sequence, thereby two different tRNA amino acid species are able to induce antitermination. Additionally, two alternative yvbW specifier codons were tested.…”

“…Folding of the yvbW leader sequence suggested a CUC leucine specifier sequence. In a recent study by Saad et al [10], the Clostridium acetobutylicum gatCAB operon was demonstrated to respond to two adjacent specifier sequence codons and two separate amino acid signals. Two additional specifier codon possibilities in the yvbW specifier loop were identified as a UGC serine codon or a GCG arginine codon.…”

“…For many T box family genes, the specificity of the amino acid signal has been determined experimentally [3] [8]- [10] or has been predicted based on the physiology of the gene; for instance tRNA synthetase genes and amino acid biosynthesis genes are regulated by their cognate amino acid class. When the amino acid class can not be determined by the physiology of the T box gene (for instance with transporters or hypothetical ORFs), the amino acid specificity can be determined by the cumulative analysis of several factors.…”

A glyS T-box riboswitch with species-specific structural features responding to both proteinogenic and nonproteinogenic tRNAGly isoacceptors

“…Moreover, T-boxes displaying SLs with relaxed codon specificity are more widespread than previously thought. It has been proposed that the specificity of the T-box for tRNA recognition depends both on the number of the nucleotides forming the SL and most importantly, on the downstream genes, which can be organized in operons and may control more than one metabolic pathway (Saad et al 2013).…”