KsgA facilitates ribosomal small subunit maturation by proofreading a key structural lesion

Abstract

Ribosome assembly is orchestrated by many assembly factors, including ribosomal RNA methyltransferases whose precise role is poorly understood. Here, we leverage the power of cryo-EM and machine learning to discover that the bacterial methyltransferase KsgA performs a novel "proofreading" function in assembly of the ribosomal small subunit by recognizing and partially disassembling particles that have matured but are not competent for translation. We propose that this activity allows inactive particles an opportunity to reassemble into an active state, thereby increasing overall assembly fidelity. Detailed structural quantifications in our datasets additionally enabled expansion of the Nomura assembly map to highlight rRNA helix and r-protein interdependencies, which newly details how binding and docking of these elements are tightly coupled. These results have wide-ranging implications in our understanding of the quality control mechanisms governing ribosome biogenesis, and showcase the power of heterogeneity analysis in cryo-EM to unveil functionally relevant information in biological systems.

Rapid structural analysis of bacterial ribosomesin situ

“…Whereas we have demonstrated the feasibility of our approach to resolve a ribosomal structure in situ with one week of processing, addressing specific biological questions will often require further specialized analyses. For example, to resolve, quantify, and spatially locate distinct functional states, one could leverage the generative capacity of tomoDRGN to reconstruct a large ensemble of heterogeneous structures to be queried on a per-particle basis for the occupancy of known structural elements with MAVEn 47,49 . Alternatively, automated (Kinman et al ., in preparation) or expert-guided inspection of such an ensemble could enable the discovery of novel structural states lost through averaging in the consensus refinement.…”

Two dynamic, N-terminal regions are required for function in Ribosomal RNA Adenine Dimethylase family members

“…However, in recent cryo-EM structures of E. coli KsgA bound to immature 30S ribosomes from the Ortega and Davis groups, a different mechanism from that in Figure 9A was proposed. It was argued that the N-terminal region of KsgA interacts with helix 24 (h24) of 16S rRNA and that this interaction keeps h24 from occluding the KsgA active site (Sun et al 2023).…”

“…1B). The current model for KsgA function is that it binds a conformation of the 30S ribosome that is off-pathway for maturation and this binding event promotes remodeling of the 30S to an on-pathway conformation (Connolly et al 2008; Sun et al 2023). Since ribosomes are able to assemble in Δ ksgA cells, methylation is not required for ribosome biogenesis but rather promotes KsgA dissociation from the ribosome once the remodeling event has occurred (Connolly et al 2008).…”

Uncovering structural ensembles from single-particle cryo-EM data using cryoDRGN

“…The development, theoretical foundations, and limitations of this work have been described previously (Zhong et al, 2019;Zhong et al, 2020). We have applied this approach to a number of publicly-available datasets, and found that cryoDRGN can uncover rare structural states in assembling bacterial ribosomes (Sun et al, 2022) and help visualize continuous conformational changes in spliceosome complexes (Zhong et al, 2020). CryoDRGN has also helped visualize a tilting motion of radial spike proteins important in dynein motors and ciliary motility (Gui et al, 2021).…”