Characterization of the Ribosome Biogenesis Landscape in E. coli Using Quantitative Mass Spectrometry

Abstract

The ribosome is an essential and highly complex biological system in all living cells. A large body of literature is available on the assembly of the ribosome in vitro, but a clear picture of this process inside the cell has yet to emerge. Here, we directly characterized in vivo ribosome assembly intermediates and associated assembly factors from wild-type E. coli cells using a general quantitative mass spectrometry (qMS) approach. The presence of distinct populations of ribosome assembly intermediates was verified using an in vivo stable isotope pulse-labeling approach, and their exact ribosomal protein (r-protein) contents were characterized against an isotopically labeled standard. The model-free clustering analysis of the resultant protein levels for the different ribosomal particles produced four 30S assembly groups that correlate very well with previous in vitro assembly studies of the small ribosomal subunit, and six 50S assembly groups that clearly define an in vivo assembly landscape for the larger ribosomal subunit. In addition, de novo proteomics identified a total of 21 known and potentially new ribosome assembly factors co-localized with various ribosomal particles. These results represent new in vivo assembly maps of the E. coli 30S and 50S subunits, and the general qMS approach should be a solid platform for future studies of ribosome biogenesis across a host of model organisms.

A hierarchical model for assembly of eukaryotic 60S ribosomal subunit domains

“…The ''body'' substructure (containing the 59 and central domains of 18S rRNA) is formed first, followed by the ''head'' substructure (containing the 39 major domain of 18S rRNA). This bipartite assembly of eukaryotic 40S subunits in vivo parallels observations from thermodynamic and kinetic studies of bacterial 30S subunit assembly in vitro and in vivo (Held et al 1974;Mulder et al 2010;Chen and Williamson 2013).…”

“…More recently, a refined in vivo assembly map was put forward by Chen and Williamson (2013) that largely corresponds to the Nierhaus map (Rohl and Nierhaus 1982). Our collective studies on yeast RPLs (Babiano et al 2012;Jakovljevic et al 2012;Gamalinda et al 2013;Ohmayer et al 2013) show the following differences from bacterial large subunit assembly: First, while incorporation of bacterial RPLs occurs via distinct assembly groups, most yeast RPLs are present in early assembly intermediates but differ in the step of assembly for which they are required and become more stably assembled in a sequential fashion.…”

“…It has become increasingly apparent that the hierarchical assembly of small ribosomal subunit domains is largely conserved from bacteria to eukaryotes (Held et al 1974;Ferreira-Cerca et al 2005Mulder et al 2010;O'Donohue et al 2010;Chen and Williamson 2013;Clatterbuck Soper et al 2013). What remained unclear is whether the same is true for large ribosomal subunits.…”

“…Assembly of both large subunits begins at the convex solvent side opposite the peptidyl transferase center and concludes with the formation of the central protuberance (Supplemental Fig. 8B; Chen and Williamson 2013;Li et al 2013). The middle step of bacterial large subunit assembly is dispersed around the 50S structure (Chen and Williamson 2013), whereas for 60S subunits, it involves the formation of the polypeptide exit tunnel.…”

“…8B; Chen and Williamson 2013;Li et al 2013). The middle step of bacterial large subunit assembly is dispersed around the 50S structure (Chen and Williamson 2013), whereas for 60S subunits, it involves the formation of the polypeptide exit tunnel. These observations suggest that the principles underlying the biosynthesis of large ribosomal subunits appear to be evolutionarily conserved despite the more numerous steps of pre-rRNA processing, additional rRNA and protein elements, and the added complexity of intranuclear trafficking and nucleocytoplasmic export in eukaryotes.…”