RNA folding pathways and the self-assembly of ribosomes

Abstract

Many RNAs do not directly code proteins but are nonetheless indispensable to cellular function. These strands fold into intricate three-dimensional shapes that are essential structures in protein synthesis, splicing, and many other processes of gene regulation and expression. A variety of biophysical and biochemical methods are now showing, in real time, how ribosomal subunits and other ribonucleoprotein complexes assemble from their molecular components. Footprinting methods are particularly useful for studying the folding of long RNAs: they provide quantitative information about the conformational state of each residue and require little material. Data from footprinting complement the global information available from small-angle X-ray scattering or cryo-electron microscopy, as well as the dynamic information derived from single-molecule Förster resonance energy transfer (FRET) and NMR methods. In this Account, I discuss how we have used hydroxyl radical footprinting and other experimental methods to study pathways of RNA folding and 30S ribosome assembly. Hydroxyl radical footprinting probes the solvent accessibility of the RNA backbone at each residue in as little as 10 ms, providing detailed views of RNA folding pathways in real time. In conjunction with other methods such as solution scattering and single-molecule FRET, time-resolved footprinting of ribozymes showed that stable domains of RNA tertiary structure fold in less than 1 s. However, the free energy landscapes for RNA folding are rugged, and individual molecules kinetically partition into folding pathways that lead through metastable intermediates, stalling the folding or assembly process. Time-resolved footprinting was used to follow the formation of tertiary structure and protein interactions in the 16S ribosomal RNA (rRNA) during the assembly of 30S ribosomes. As previously observed in much simpler ribozymes, assembly occurs in stages, with individual molecules taking different routes to the final complex. Interactions occur concurrently in all domains of the 16S rRNA, and multistage protection of binding sites of individual proteins suggests that initial encounter complexes between the rRNA and ribosomal proteins are remodeled during assembly. Equilibrium footprinting experiments showed that one primary binding protein was sufficient to stabilize the tertiary structure of the entire 16S 5'-domain. The rich detail available from the footprinting data showed that the secondary assembly protein S16 suppresses non-native structures in the 16S 5'-domain. In doing so, S16 enables a conformational switch distant from its own binding site, which may play a role in establishing interactions with other domains of the 30S subunit. Together, the footprinting results show how protein-induced changes in RNA structure are communicated over long distances, ensuring cooperative assembly of even very large RNA-protein complexes such as the ribosome.

Figure 1. Hydroxyl radical footprinting of RNA and protein interactions

Ribose sugars buried by RNA tertiary interactions or protein contacts are protected from attack by hydroxyl radical, producing a gap or “footprint” in the distribution of cleaved products. Radicals are produced by the Fenton reaction or X-ray photolysis of water; strand scission products are analyzed and quantified by primer extension with reverse transcriptase. Base oxidation products are not readily detected by this method.

Figure 2. Folding pathway of the Tetrahymena ribozyme

Time-resolved footprinting^, showed that tertiary interactions in the P4–P6 domain (green) form in ~1 s, more rapidly than contacts in peripheral helices (pink and grey) and in the P3–P9 domain (blue), due to mispairing of the P3 helix (gold). The ensemble of unfolded structures (U) partitions among parallel pathways, with 5–10% of molecules folding directly to the native state (N).

Figure 3. X-ray footprinting of 30S ribosome assembly

The evolution of RNA and RNA-protein interactions was probed from 20 ms to 120 s, using a 10 ms X-ray pulse. Native E. coli 16S rRNA was pre-folded in reconstitution buffer before addition of native E. coli 30S proteins (TP30). Quantitation of primer extension products revealed multi-stage folding of individual residues throughout the 16S rRNA.

Figure 4. Induced fit in rRNA-protein interactions

Different residues in a single 30S protein binding site are protected with different rate constants, when probed by X-ray footprinting. (a) Residues contacted by protein S4 in mature 30S subunits colored according to the rate of protection: red, ≥ 20s⁻¹; orange, 2–20 s⁻¹; green, 0.2 –2 s⁻¹; blue, 0.01 – 0.2 s⁻¹. (b) Residues contacted by protein S16, colored as in (a). Reproduced from Ref. with permission.

Figure 5. S16-dependent conformational switch

(a) Proteins that bind the 16S 5′ domain (body) stabilize RNA tertiary interactions, lowering the Mg²⁺ concentration required for protection from hydroxyl radical cleavage.^, Fitted curves for protection of residues in helix 15 (nt 378; dashed lines) and helix 18 (nt 501–502; solid lines) in the presence of S4, S17 and S20, without S16 (pink) or with S16 (blue). (b) Exposure of helix 18 can be explained by movement of helix 3 (purple) during assembly. Ribbon (pdb 2avy) shows 16S nt 24–562 with proteins as pastel surfaces; S4 (pink), S17 (green), S20 (yellow), S16 (blue). (c) Minimal model for 5′ domain assembly, involving intermediate RNPs with native (I_N) or non-native (I_nC) configuration in the lower half of the domain near helices 6, 6a, 10, 15. Helix 3 is displaced in I_N, then moves back into position in the native complex (N). S16 smooths the path of assembly by favoring I_N over I_nC, and by stabilizing N. Reproduced from Ref. with permission.