Two-codon T-box riboswitch binding two tRNAs

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• T-box riboswitches regulate gene transcription via tRNA binding and terminator/antiterminator structure formation.
• Previously, T-boxes were considered single-specific, binding only one tRNA anticodon through their specifier loop (SL).

Purpose of the Study:

• To investigate the specificity of a T-box riboswitch in Clostridium acetobutylicum that regulates an essential tRNA-dependent transamidation pathway.
• To determine if this T-box exhibits dual specificity due to overlapping codons in its SL for tRNA(Asn) and tRNA(Glu).

Main Methods:

• Extensive mutagenic analyses
• Biochemical assays
• Chemical probing experiments
• Genomic comparisons

Main Results:

• The T-box riboswitch demonstrated efficient binding of both tRNA(Asn) and tRNA(Glu) both in vitro and in vivo.
• Dual specificity is achieved through a single base shift in the SL, enabling recognition of overlapping codons.
• Genomic data suggest widespread occurrence of such flexible T-boxes in bacteria, with specificity linked to metabolic pathway interconnections.

Conclusions:

• T-box riboswitches can exhibit dual specificity, sensing multiple tRNAs via overlapping codons in the SL.
• This mechanism allows for balanced biosynthesis of multiple amino acids, representing a unique transcriptional regulation strategy.
• The codon ambiguity of SLs is genome-dependent, highlighting metabolic context in riboswitch evolution.