Assembly of the 30s Ribosome From the RNA Folding Perspective

Abstract

Ribosome assembly requires folding of the rRNA and the hierarchical addition of 20 or more proteins to the complex. We visualized assembly of the bacterial 30S ribosomal subunit in real time using time-resolved hydroxyl radical footprinting. This method reveals the extent of RNA and protein interactions at each segment of the RNA backbone, providing a detailed view of the changes to the rRNA structure during assembly. Each domain of the 30S ribosome assembles concurrently in vitro, and many tertiary RNA interactions and RNA-protein interactions are established within the first 0.1 seconds. Individual proteins protect different segments of their binding site at different rates, suggesting that the initial protein-RNA complexes are remodeled during assembly. By perturbing the free energy of RNA-protein complexes from the body of the 30S subunit, we find that a single protein can stabilize an entire domain of the 16S rRNA. However, multiple proteins bound to the same domain narrow the ensemble of rRNA conformations. Specific structural switches stabilize the decoding active site and enable long-range structural communication within the 30S ribosomal subunit.