Anti-sense oligonucleotide probing as a structural platform for studying ribonucleoprotein complex assembly

Area of Science:

• Structural biology
• Molecular biology
• Biochemistry

Background:

• Investigating the folding kinetics of large RNA-protein complexes (RNPs), such as the bacterial ribosome, is challenging.
• Previous methods relied on genetic approaches to modulate protein or factor expression.

Purpose of the Study:

• To probe RNP assembly by generating novel folding intermediates.
• To elucidate the hierarchical structure and dynamics of RNP assembly.

Main Methods:

• Utilized anti-sense oligonucleotides (ASOs) to disrupt RNA/RNA and RNA/protein interactions.
• Employed an in vitro co-transcriptional ribosome assembly assay.
• Determined intermediate structures using cryo-electron microscopy (cryo-EM).

Main Results:

• Identified 10 assembly inhibitor ASOs and determined 38 intermediate structures using cryo-EM.
• Provided evidence for independent rRNA domain folding prior to interdomain docking.
• Discovered that PNAs targeting 23S rRNA domain-I subdivide the assembly core into smaller blocks.

Conclusions:

• Developed an assembly graph revealing template-directed RNA docking (foldons) and domain consolidation.
• Established a hierarchical model for RNP assembly.
• Identified potential antibiotic targets and a platform for studying RNP structure and dynamics.