New structural and functional insights into late maturation of the large ribosomal subunit in Escherichia coli

Abstract

For more than six decades, the ribosome has been subject of intensive research, addressing predominantly its essential function in protein synthesis. However, considerably less is known about how ribosomes assemble from their individual components and how ribosome assembly is coordinated and facilitated by enzymatic assembly factors in vivo. Recent advances in both fluorescence microscopy and cryogenic electron microscopy (cryo-EM) now offer tools to address the mechanistic details of this multilayered process. This work focusses on late processes during the assembly of the large 50S subunit of Escherichia coli (E. coli) and provides a systematic, functional analysis of the late acting assembly factors DbpA, EngA, EngB, RlmE and ObgE. An RNA-antisense-based study was employed to systematically characterize the consequences of assembly factor depletion. It was found that the absence of each of the five investigated factors caused mild to severe growth perturbation at different temperatures and an accumulation of pre-50S or 50S particles, emphasizing the central role of these five assembly factors in correct 50S maturation. Moreover, a super-resolution fluorescence microscopic technique based on 3D-SIM was established and revealed that assembly defective cells exhibit a specific morphological phenotype resulting in a considerable reorganization of ribosomal particles, reminiscent of cells treated with translation inhibitors. This detailed phenotypic and morphological characterization was complement-ed by an approach utilizing engineered E. coli strains harboring assembly factors fused with epitope tags enabling immunodetection or purification of bona fide ribosomal precursors for subsequent structural analyses. As proof of concept, the E. coli strain harboring StrepII-tagged GTPase ObgE was used to isolate and structurally analyze native pre-50S•ObgE-StrepII complexes by cryo-EM to elaborate ObgE´s contribution to final pre-50S assembly. The data suggest a gatekeeper function for ObgE by sensing critical conformational changes within the large subunit´s active site region that ultimately trigger GTP hydrolysis and ObgE dissociation. In addition, this study provides ten phenotypically validated strains (five knock-down and five knock-in strains, respectively) as toolbox for further structural and morphological analyses of late 50S assembly.