A pseudouridine synthase shapes tRNA structural dynamics through both catalysis and remodeling.

Area of Science:

• Molecular biology
• Biochemistry
• Structural biology

Background:

• Transfer RNA (tRNA) is a crucial molecule known for its stability and structure.
• tRNA undergoes significant structural alterations when interacting with modification enzymes.
• Understanding these dynamics is key to tRNA maturation and function.

Purpose of the Study:

• To investigate the structural dynamics of tRNA before and after interaction with the pseudouridine synthase Pus4/TruB.
• To elucidate the role of pseudouridylation in tRNA conformation and Pus4/TruB binding.
• To compare the effects of wild-type Pus4/TruB and a catalytically inactive mutant on tRNA structure.

Main Methods:

• Optical binding assays were employed to study molecular interactions.
• Single-molecule Förster Resonance Energy Transfer (smFRET) was used to observe tRNA structural dynamics.
• Conformational states of tRNA were analyzed in the presence and absence of Pus4/TruB.

Main Results:

• Unmodified and pseudouridylated tRNA dynamically sample open and closed conformations.
• Pus4/TruB binding to unmodified tRNA induces additional conformational states.
• Pre-pseudouridylated tRNA approaches the final conformational ensemble more rapidly upon Pus4/TruB engagement.
• A catalytically inactive Pus4/TruB mutant exhibits slower binding and induces distinct conformational ensembles compared to the wild-type enzyme.

Conclusions:

• Pus4/TruB catalyzes a chemical modification on tRNA and induces lasting structural changes.
• The enzyme's interaction with tRNA is dynamic and influences subsequent tRNA maturation steps.
• Pseudouridylation plays a significant role in modulating tRNA conformation and Pus4/TruB activity.