Abstract
Assembly of 30S ribosomes involves the hierarchical addition of ribosomal proteins that progressively stabilize the folded 16S rRNA. Here, we use three-color single molecule FRET to show how combinations of ribosomal proteins uS4, uS17 and bS20 in the 16S 5′ domain enable the recruitment of protein bS16, the next protein to join the complex. Analysis of real-time bS16 binding events shows that bS16 binds both native and non-native forms of the rRNA. The native rRNA conformation is increasingly favored after bS16 binds, explaining how bS16 drives later steps of 30S assembly. Chemical footprinting and molecular dynamics simulations show that each ribosomal protein switches the 16S conformation and dampens fluctuations at the interface between rRNA subdomains where bS16 binds. The results suggest that specific protein-induced changes in the rRNA dynamics underlie the hierarchy of 30S assembly and simplify the search for the native ribosome structure.
Highlights
Of 30S ribosomes involves the hierarchical addition of ribosomal proteins that progressively stabilize the folded 16S rRNA
Structural and biophysical studies showed that protein uS15 preferentially binds the folded conformation of a three-helix junction in the 16S central domain4, 5. uS15 binding pre-organizes an adjacent helix junction[6], lowering the entropic penalty for binding the proteins in the assembly map[7, 8]
After 1–2 s, the S4-rRNA complexes reach a slow dynamic equilibrium between the flipped intermediate complex and the native complex, in which h3 is docked against S4 as observed in the mature ribosome (Fig. 1a)
Summary
Of 30S ribosomes involves the hierarchical addition of ribosomal proteins that progressively stabilize the folded 16S rRNA. US15 binding pre-organizes an adjacent helix junction[6], lowering the entropic penalty for binding the proteins in the assembly map[7, 8] Such “progressive stabilization” models explain why protein binding stabilizes the rRNA in its native conformation, certain ribosomal proteins, such as uS4 and bS16, are indispensable for assembly[9, 10] even in Mg2+ concentrations sufficient to fold the rRNA in the absence of protein[11]. We use three-color single-molecule FRET19 to directly observe the binding of multiple ribosomal proteins to the rRNA, and investigate the physical origins of cooperative assembly
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