Lineage-specific insertions in T-box riboswitches modulate antibiotic binding and action

Area of Science:

• Molecular Biology
• RNA Biology
• Microbial Genetics

Background:

• T-box riboswitches regulate gene expression in bacteria, particularly in staphylococci.
• The glyS T-box in staphylococci features a unique stem Sa insertion in its regulatory domain.
• Stem Sa in Staphylococcus aureus T-boxes interacts with antibiotics, influencing transcription.

Purpose of the Study:

• To investigate the functional role of the stem Sa insertion in staphylococcal glyS T-boxes.
• To determine how stem Sa affects transcription readthrough and tRNA isoacceptor discrimination.
• To explore the potential of stem Sa as a species-selective drug target.

Main Methods:

• Domain swap mutagenesis was employed to analyze stem Sa function.
• Probing analysis was used to assess structural and functional impacts.
• In vitro and in vivo transcription assays were performed.

Main Results:

• Deletion of stem Sa significantly reduced S. aureus glyS T-box transcription and tRNA discrimination.
• Antibiotic effects on transcription were inverted upon stem Sa deletion.
• Insertion of stem Sa into a G. kaustophilus T-box enhanced transcription with tigecycline and improved tRNA discrimination.

Conclusions:

• Stem Sa is essential for efficient staphylococcal glyS T-box-mediated transcription and tRNA recognition.
• Stem Sa's antibiotic-binding capability makes it a potential target for developing species-selective antibiotics.