Ribosome RNA assembly intermediates visualized in living cells

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Cellular RNAs, like 16S rRNA, exist in dynamic conformational ensembles before forming functional complexes.
• Understanding in-cell RNA structures is crucial for elucidating complex biological processes such as protein synthesis.

Purpose of the Study:

• To investigate the in-cell structure of Escherichia coli 16S rRNA during active growth.
• To identify factors influencing RNA conformational ensembles within living bacterial cells.

Main Methods:

• Utilized single-nucleotide resolution selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to probe RNA structure in vivo.
• Employed SHAPE-directed modeling and in-cell RNA structure pulse-chase experiments to analyze conformational dynamics.

Main Results:

• The predominant 16S rRNA structure in growing cells differed from structures predicted by comparative sequence analysis and high-resolution studies.
• Halting transcription induced a structural shift in 16S rRNA towards the expected conformation.
• Alternate RNA conformations under growth conditions involve interactions with ribosomal proteins, forming a C-shape around the mRNA channel.

Conclusions:

• Ribosome assembly factors act as molecular scaffolds, preorganizing RNA intermediates.
• The final stages of ribonucleoprotein assembly involve significant protein-mediated RNA conformational rearrangements.