Scaffold-enabled high-resolution cryo-EM structure determination of RNA.

Area of Science:

• Structural Biology
• Molecular Biology
• Biochemistry

Background:

• Determining high-resolution structures of protein-free RNAs, especially small ones, using cryo-electron microscopy (cryo-EM) is challenging due to low molecular weight and limited nucleotide-level detail.
• Previous cryo-EM studies often resulted in low to moderate resolution, hindering a comprehensive understanding of RNA structure-function relationships.

Purpose of the Study:

• To develop and demonstrate a novel strategy for high-resolution cryo-EM structure determination of small RNAs.
• To apply this method to elucidate the structures of the thiamine pyrophosphate (TPP) riboswitch aptamer domain and the bacterial non-coding RNA raiA.

Main Methods:

• A scaffolding technique involving the fusion of small RNAs to a group II intron was employed.
• Cryo-EM was utilized to determine the high-resolution structures of the fused RNA complexes.
• The method was validated by analyzing the thiamine pyrophosphate (TPP) riboswitch aptamer and the raiA RNA.

Main Results:

• The scaffolding approach successfully yielded high-resolution cryo-EM structures of the appended RNAs.
• The structure of the TPP riboswitch aptamer domain was determined at 2.5 Å resolution, revealing the ligand binding pocket.
• The ligand-free apo state of the TPP riboswitch showed an open Y-shaped conformation, and the raiA RNA structure was also resolved at 2.5 Å.

Conclusions:

• The developed RNA scaffolding strategy significantly enhances the capability of cryo-EM for determining high-resolution structures of small and previously intractable RNAs.
• This versatile method provides unprecedented structural insights into key RNA molecules like the TPP riboswitch and raiA, facilitating further functional studies.