Single-molecule visualization of sequence-specific RNA binding by a designer PPR protein

Area of Science:

• Molecular Biology
• Biochemistry
• Biotechnology

Background:

• Pentatricopeptide repeat (PPR) proteins are modular RNA-binding proteins.
• Designer PPRs (dPPRs) offer potential in diagnostics and RNA localization.
• The mechanism of PPR-RNA sequence recognition remains unclear.

Purpose of the Study:

• To elucidate the real-time mechanism of single-stranded RNA (ssRNA) binding by dPPRs.
• To determine the structural basis of dPPR-target ssRNA interactions.
• To investigate factors influencing dPPR binding efficiency and specificity.

Main Methods:

• X-ray crystallography to determine dPPR-ssRNA complex structure.
• Single-molecule fluorescence resonance energy transfer (smFRET) for real-time binding kinetics.
• Investigated binding to ssRNA sequences of varying lengths and structures.

Main Results:

• dPPRs exhibit slower binding to longer ssRNA sequences, or fail to bind.
• Stable secondary structures in ssRNA sequester target sites, hindering dPPR binding.
• dPPRs bind exclusively to their cognate target sequence and do not scan longer oligonucleotides.
• Binding is limited by 3D diffusion kinetics.

Conclusions:

• dPPR binding efficiency is constrained by target ssRNA length and secondary structure.
• The lack of scanning and diffusion limitations may explain PPR protein localization in organelles.
• Understanding these mechanisms is crucial for dPPR-based biotechnological applications.