Near-physiological in vitro assembly of 50S ribosomes involves parallel pathways

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Understanding macromolecular complex assembly is crucial but challenging.
• Ribosomes, as ribonucleoprotein complexes, are model systems for studying assembly.
• The assembly process of the large ribosomal subunit (50S) is complex and not fully understood.

Purpose of the Study:

• To investigate the intermediate structures during the in vitro assembly of the 50S ribosomal subunit.
• To elucidate the principles and pathways governing 50S subunit assembly.

Main Methods:

• Utilized a near-physiological, co-transcriptional in vitro reconstitution system.
• Employed cryo-electron microscopy (cryo-EM) single-particle analysis.
• Applied heterogeneous subclassification to resolve thirteen distinct pre-50S intermediate structures.

Main Results:

• Resolved thirteen cryo-EM maps detailing the 50S subunit assembly process.
• Identified fourteen cooperative assembly blocks that contribute to 50S subunit formation.
• Discovered the smallest reported assembly core, comprising folded rRNA and proteins, serving as a foundation for assembly.

Conclusions:

• The 50S ribosomal subunit assembles through a series of defined, cooperative steps involving fourteen distinct blocks.
• Assembly follows specific dependencies, indicating parallel pathways during both early and late stages.
• This work provides a detailed structural framework for understanding ribosome biogenesis.