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.
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.
|Duss, Olivier; Stepanyuk, Galina A; Grot, Annette et al. (2018) Real-time assembly of ribonucleoprotein complexes on nascent RNA transcripts. Nat Commun 9:5087|
|Solis, Gregory M; Kardakaris, Rozina; Valentine, Elizabeth R et al. (2018) Translation attenuation by minocycline enhances longevity and proteostasis in old post-stress-responsive organisms. Elife 7:|
|Tan, Yong Zi; Baldwin, Philip R; Davis, Joseph H et al. (2017) Addressing preferred specimen orientation in single-particle cryo-EM through tilting. Nat Methods 14:793-796|
|Davis, Joseph H; Tan, Yong Zi; Carragher, Bridget et al. (2016) Modular Assembly of the Bacterial Large Ribosomal Subunit. Cell 167:1610-1622.e15|
|Dai, Xiongfeng; Zhu, Manlu; Warren, Mya et al. (2016) Reduction of translating ribosomes enables Escherichia coli to maintain elongation rates during slow growth. Nat Microbiol 2:16231|
|Lavergne, Thomas; Lamichhane, Rajan; Malyshev, Denis A et al. (2016) FRET Characterization of Complex Conformational Changes in a Large 16S Ribosomal RNA Fragment Site-Specifically Labeled Using Unnatural Base Pairs. ACS Chem Biol 11:1347-53|
|Earnest, Tyler M; Lai, Jonathan; Chen, Ke et al. (2015) Toward a Whole-Cell Model of Ribosome Biogenesis: Kinetic Modeling of SSU Assembly. Biophys J 109:1117-35|
|Gomez-Amaro, Rafael L; Valentine, Elizabeth R; Carretero, Maria et al. (2015) Measuring Food Intake and Nutrient Absorption in Caenorhabditis elegans. Genetics 200:443-54|
|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|
|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|
Showing the most recent 10 out of 37 publications