Structural Visualization of the Formation and Activation of the 50S Ribosomal Subunit during In Vitro Reconstitution

Abstract

The assembly of ribosomal subunits is an essential prerequisite for protein biosynthesis in all domains of life. Although biochemical and biophysical approaches have advanced our understanding of ribosome assembly, our mechanistic comprehension of this process is still limited. Here, we perform an in vitro reconstitution of the Escherichia coli 50S ribosomal subunit. Late reconstitution products were subjected to high-resolution cryo-electron microscopy and multiparticle refinement analysis to reconstruct five distinct precursors of the 50S subunit with 4.3-3.8 Å resolution. These assembly intermediates define a progressive maturation pathway culminating in a late assembly particle, whose structure is more than 96% identical to a mature 50S subunit. Our structures monitor the formation and stabilization of structural elements in a nascent particle in unprecedented detail and identify the maturation of the rRNA-based peptidyl transferase center as the final critical step along the 50S assembly pathway.

Defects in the assembly of ribosomes selected for β-amino acid incorporation

“…We were able to detect stoichiometric levels of RPs uL6, uL10, and uL11, whose cryo-EM density is often weak due to motion in the large subunit arms. 35,37 In the vicinity of the P7A7 mutations in the PTC, we observe a stark contrast between well-ordered regions seen in the WT structure, with features like base stacking clearly resolved, and adjacent regions where the density is mainly noise (Figure 4). Comparison of these disordered regions in the P7A7 50S subunit map with the corresponding region in the WT PTC shows several interactions that would be perturbed by some of the mutations (Figure 5) , 38 and may be responsible for the structural defects we observe.…”

“…Heterogeneous ab initio reconstructions of the P7A7 50S subunit revealed two classes of particles that represent the large subunit with two different types of disorder. The major class lacks density for ribosomal proteins uL6, uL10, uL11, uL16, bL33, and bL35 (excluding proteins bL31 and bL36 which are known to disassociate during ribosome isolation), 35 as well as multiple stretches of the 23S rRNA comprising the PTC, helices H44, H71, H89-H93, and the end of H39. The smaller class further lacks the central protuberance entirely ( Figure S4 for workflow).…”

“…This connectivity is strikingly similar to the non-native PTC organization seen in late intermediates of the 50S assembly pathway both in vivo and vitro . 35,37 While ribosome biogenesis is a complex process, a general order of rRNA helix formation and ribosomal protein association is known. 39,40 Despite multiple assembly routes, resulting in a spectrum of assembly intermediates, he PTC is usually observed as the last-assembling 50S motif.…”

“…To further explore the relationship of the observed P7A7 structure with assembly pathways, we carried out a detailed tabulation of structural features across the P7A7 and WT 50S subunit EM maps and compared them to known assembly intermediates, including states IV and V from characterization of in vitro reconstitution 35 and states E4 and E5 from in vivo intermediates caused by bL17 knockdown. 37 Using a previously developed calculation method 37 Nevertheless, the large-scale organization of several intermediates is quite similar despite the different methods of assembly perturbation, suggesting that a PTC folding defect is a common roadblock in 50S ribosomal subunit maturation.…”

“…Of particular note are two chaperones, ObgE and EngA, that are proposed to interact with the PTC during assembly and may help direct PTC folding away from inactive intermediates, such as the non-native fold seen in P7A7 ( Figure 5) . 35,57,58 Thus, due to an unstable PTC, P7A7 ribosomes are not suited for detailed in vitro study of improved β-amino acid polymerization reactions by the ribosome. Future efforts toward an understanding could include optimizing a cytosol-mimicking buffer for purification and analysis or mapping in vivo RNA structure of P7A7 ribosomes to assess whether it possesses a more stable PTC in cells.…”