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

The ribosome is an essential and highly complex biological system in all living cells. A large body of literature on the assembly of the ribosome in vitro is available, 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 Escherichia 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 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 prove to be a solid platform for future studies of ribosome biogenesis across a host of model organisms.

Graphical AbstractFigure optionsDownload high-quality image (73 K)Download as PowerPoint slideHighlights
► The assembly of ribosomes inside living cells is relatively unexplored.
► In vivo ribosome assembly intermediates were directly characterized by qMS.
► Clustering analysis reveals new 30S and 50S in vivo assembly maps.
► Proteomics identify both known and possibly novel ribosome assembly factors.
► This qMS approach should open new horizons in the future study of ribosomes.