Dynamics of the Bacterial Ribosome and Proteome

Project Summary This application describes a broadly based biophysical characterization of the process of ribosome assembly in bacteria. Ribosomes are the essential machines in all cells that synthesize proteins, and in bacteria, ribosome synthesis accounts for about one third of the energy expenditure for cell division. Ribosome biogenesis is highly efficient, involving assembly of ~50 RNA and protein components, with the assistance of ~ 50 assembly factors. This process will be will be investigated in vitro and in bactrerial cells using single particle electron microscopy, mass spectrometry, cryo-electron tomography light microscopy. Electron microscopy of intermediates that accumulate under perturbed conditions has revealed a distribution of structures that can be ordered into a putative assembly pathway. A current focus is to understand the role of the assembly factors in promoting efficient ribosome biogenesis. In addition, we are investigating the inhibition of ribosome assembly both in vitro using nucleic acid hybridization, and in cells using CRISPR RNAs. Finally, we will characterize the locus of ribosome assembly in cells using cryo-electron tomography to obtain structure and localization of intermediates, and using MINFLUX nanoscopy to obtain nanometer scale information on the ultrastructure of the heavily transcribed ribosomal operon. In the coming period, we hope to merge our molecular understanding from in vitro studies with a physiological understanding of the central role of ribosome biogenesis in bacteria.