This proposal outlines a plan to investigate the mechanism of assembly of E. coli ribosomes both in vitro and in vivo. The ribosome is the macromolecular machine responsible fbr all protein synthesis in cells, and it is efficiently assembled from over 50 components. Using a combination of biophysical methods, we will Investigate the order of events in assembly and the kinetics of assembly in vitro. We will examine the role of assembly cofactors in E. coli, and examine the effects of antibiotics on the assembly process. We have developed a novel isotope pulse-chase assay that enables measurement of binding kinetics for ribosomal proteins using mass spectrometry. We are developing a two-photon fluorescence correlation microscope to monitor the assembly of fluorescently labeled ribosomal proteins in real time. Finally, we are extending our mass spectrometry analysis to isotope pulse experiments in living bacteria, which allows us to directly monitor the biogenesis of ribosomes. Taken together, these studies will provide new mechanistic insights into the critical process of ribosome assembly.

Public Health Relevance

The assembly of ribosomes is essential for protein synthesis In all cells. The proposed studies will help elucidate the understanding of ribosome biogenesis in bacteria. Resulting insights may lead to novel anti-infective strategies.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (NSS)
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Flicker, Paula F
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Scripps Research Institute
La Jolla
United States
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Popova, Anna M; Williamson, James R (2014) Quantitative analysis of rRNA modifications using stable isotope labeling and mass spectrometry. J Am Chem Soc 136:2058-69
Ban, Nenad; Beckmann, Roland; Cate, Jamie H D et al. (2014) A new system for naming ribosomal proteins. Curr Opin Struct Biol 24:165-9
Stokes, Jonathan M; Davis, Joseph H; Mangat, Chand S et al. (2014) Discovery of a small molecule that inhibits bacterial ribosome biogenesis. Elife 3:e03574
Gulati, Megha; Jain, Nikhil; Davis, Joseph H et al. (2014) Functional interaction between ribosomal protein L6 and RbgA during ribosome assembly. PLoS Genet 10:e1004694
Jomaa, Ahmad; Jain, Nikhil; Davis, Joseph H et al. (2014) Functional domains of the 50S subunit mature late in the assembly process. Nucleic Acids Res 42:3419-35
Sashital, Dipali G; Greeman, Candacia A; Lyumkis, Dmitry et al. (2014) A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30S subunit assembly in E. coli. Elife 3:
Chen, Stephen S; Williamson, James R (2013) Characterization of the ribosome biogenesis landscape in E. coli using quantitative mass spectrometry. J Mol Biol 425:767-79
Ridgeway, William K; Millar, David P; Williamson, James R (2012) The spectroscopic basis of fluorescence triple correlation spectroscopy. J Phys Chem B 116:1908-19
Menichelli, Elena; Edgcomb, Stephen P; Recht, Michael I et al. (2012) The structure of Aquifex aeolicus ribosomal protein S8 reveals a unique subdomain that contributes to an extremely tight association with 16S rRNA. J Mol Biol 415:489-502
Shoji, Shinichiro; Dambacher, Corey M; Shajani, Zahra et al. (2011) Systematic chromosomal deletion of bacterial ribosomal protein genes. J Mol Biol 413:751-61

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