Measuring the dynamics of E. coli ribosome biogenesis using pulse-labeling and quantitative mass spectrometry

Mol Biosyst. 2012 Oct 30;8(12):3325-34. doi: 10.1039/c2mb25310k.

Abstract

The ribosome is an essential organelle responsible for cellular protein synthesis. Until recently, the study of ribosome assembly has been largely limited to in vitro assays, with few attempts to reconcile these results with the more complex ribosome biogenesis process inside the living cell. Here, we characterize the ribosome synthesis and assembly pathway for each of the E. coli ribosomal protein (r-protein) in vivo using a stable isotope pulse-labeling timecourse. Isotope incorporation into assembled ribosomes was measured by quantitative mass spectrometry (qMS) and fit using steady-state flux models. Most r-proteins exhibit precursor pools ranging in size from 0% to 7% of completed ribosomes, and the sizes of these individual r-protein pools correlate well with the order of r-protein binding in vitro. Additionally, we observe anomalously large precursor pools for specific r-proteins with known extra-ribosomal functions, as well as three r-proteins that apparently turnover during steady-state growth. Taken together, this highly precise, time-dependent proteomic qMS approach should prove useful in future studies of ribosome biogenesis and could be easily extended to explore other complex biological processes in a cellular context.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / analysis
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Mass Spectrometry
  • Nitrogen Isotopes
  • Proteomics
  • RNA, Ribosomal / chemistry
  • RNA, Ribosomal / genetics
  • RNA, Ribosomal / metabolism
  • Ribosomal Proteins / chemistry
  • Ribosomal Proteins / metabolism*
  • Ribosome Subunits, Large, Bacterial / metabolism*
  • Ribosome Subunits, Small, Bacterial / metabolism*
  • Ribosomes / metabolism*

Substances

  • Escherichia coli Proteins
  • Nitrogen Isotopes
  • RNA, Ribosomal
  • Ribosomal Proteins