RNA folding and ribosome assembly

Abstract

Ribosome synthesis is a tightly regulated process that is crucial for cell survival. Chemical footprinting, mass spectrometry, and cryo-electron microscopy are revealing how these complex cellular machines are assembled. Rapid folding of the rRNA provides a platform for protein-induced assembly of the bacterial 30S ribosome. Multiple assembly pathways increase the flexibility of the assembly process, while accessory factors and modification enzymes chaperone the late stages of assembly and control the quality of the mature subunits.

Figure 1. Folding of the 16S rRNA 5’ domain without proteins

The tertiary structure of the rRNA (nt 21-562) was probed by hydroxyl radical footprinting [15]. a. At equilibrium, the interactions were stable in 120 mM NH₄Cl (blue), low MgCl₂ ([Mg²⁺]_1/2 = 0-2.5 mM; purple), or high MgCl₂ ([Mg²⁺]_1/2 = 4-8 mM; green). b. In 20 mM MgCl₂ at 37 °C, many interactions form 60-90% in 20 ms (blue); the lower subdomain folds 40-50% in 20 ms and 30-60% in 30-60 s (orange).

Figure 2. Kinetics of 30S assembly by pulse-chase mass spectrometry

a. Nomura assembly map is colored by the protein binding rates at 37 °C: red, • 20 min^-1; orange, 8.1-15 min^-1; green, 1.2-2.2 min^-1; blue, 0.38-0.73 min^-1; purple, 0.18-0.26 min^-1. b. 30S subunit from T. thermophilus [20], colored as in a. Reprinted from [33] with permission.

Figure 3. Structural changes in the 16S rRNA during late steps of assembly

For structure mapping, reconstitution intermediate (RI) was formed from 16S rRNA plus recombinant S4-S9, S11-S13, S15-S20 at 4 °C, then shifted to 42 °C (RI*), and chased into 30S complexes with the addition of tertiary assembly proteins (S3, S3, S10, S14, S21) [36]. b. Structural differences between RI and RI* determined by dimethylsulfate base modification (red) and hydroxyl radical cleavage (light blue) [36]. N6,N6-dimethyl A1518 and A1519 products of KsgA are shown in green; blue triangles indicate ΔrimM suppressors [38].

Figure 4. Binding sites of RbfA and Era on the 30S ribosome

a. Superposition of cryo-EM reconstructions showing the locations of RbfA (red) [40] and Era (magenta) [41]. b. Both assembly factors interact with helix 28 (green) of the 16S rRNA. Models were obtained by docking the crystal structure of the 30S subunit [20] into the cryo-EM density map. Reprinted from [40] with permission.