Crystal structure of pseudouridine synthase RluA: indirect sequence readout through protein-induced RNA structure

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• RluA is a crucial enzyme catalyzing pseudouridylation, a modification of 23S ribosomal RNA (rRNA) and transfer RNAs (tRNAs).
• Pseudouridylation plays a vital role in RNA structure and function, impacting processes like protein synthesis.

Purpose of the Study:

• To elucidate the structural mechanism by which RluA recognizes and modifies its RNA substrates.
• To understand the role of enzyme-induced RNA reorganization in substrate binding and catalysis.

Main Methods:

• X-ray crystallography was employed to determine the 2.05 Å resolution structure of RluA bound to a tRNA(Phe) anticodon stem loop.
• Structure-guided mutagenesis was used to investigate the function of key residues, particularly arginine, in substrate recognition and modification.
• Sequence and structural comparisons were made with related pseudouridine synthases (RsuA, TruA).

Main Results:

• RluA binding induces a significant structural change in the tRNA anticodon loop, deviating from its canonical 'U' turn conformation.
• The modified loop adopts a novel structure featuring a reverse-Hoogsteen base pair and three flipped-out nucleotides.
• An arginine residue within RluA intercalates into the RNA structure, facilitating the flipping of the target nucleotide into the enzyme's active site.

Conclusions:

• RluA recognizes RNA substrates by probing for their ability to undergo a specific loop reorganization, rather than direct sequence recognition.
• A conserved arginine residue is critical for the base-flipping mechanism across RluA, RsuA, and TruA pseudouridine synthase families.
• The findings provide a detailed mechanistic insight into RNA pseudouridylation and enzyme-substrate interactions.