The overall goal of this project is to understand the structural basis for specific interaction S15 and 16S rRNA. We will begin by defining the minimal protein and RNA requirements for complex formation using components from B. stearothermophilus. We will characterize the structure of the free protein, free RNA, and of the S15-rRNA complex using NMR spectroscopy, and fluorescence energy transfer. We hope to learn about how the 16S RNA structure contributes to the recognition of S15, and how the binding of S15 organizes the rRNA such that ribosomal assembly can proceed.
The specific Aims are as follows: 1. Determine the minimal rRNA binding site for the S15 protein. We will begin by identifying the minimal 16S RNA fragment that completely contains the binding site for S15. 2. Structural studies of the S15 protein and protein fragments. We will characterize the free protein using NMR spectroscopy as a necessary step for studying the RNA-protein complex. 3. Prepare deuterated ribonucleotides designed for NMR of large RNAs. In order to facilitate the NMR studies of large RNAs and RNA-protein complexes, we will prepare ribonucleotides where the 3', 4', 5', and 5"""""""" proteins have been replaced by deuterons. 4. Structural studies of the free minimal rRNA fragment. We will study the structure of the minimal rRNA binding site using NMR spectroscopy. 5. Characterization of the rRNA conformational change upon S15 binding using fluorescence energy transfer. Binding of S15 may be accompanied by a conformational change in the rRNA. We will construct RNA fragments composed of multiple RNA strands that are fluorescently labeled at the 3'ends to estimate the end to end distances before and after protein binding. 6. Structural studies of the minimal S15-rRNA complex. We will characterize the structure of the minimal S15- minimal 16S RNA complex using multidimensional NMR spectroscopy. We are interested in how the RNA structure contributes to the binding, the nature of the RNA-protein contacts, and the nature of the protein-induced conformational change. 7. Characterize the subsequent binding of S6 and S18, as a model for ribosomal assembly. Beginning with the S15-rRNA complex, we will determine the minimal rRNA fragment competent to bind these three proteins, focusing on the additional interactions formed by S6/S18.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Scripps Research Institute
La Jolla
United States
Zip Code
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:
Jin, Hyun Yong; Oda, Hiroyo; Chen, Pengda et al. (2017) Differential Sensitivity of Target Genes to Translational Repression by miR-17~92. PLoS Genet 13:e1006623
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
Ridgeway, William K; Millar, David P; Williamson, James R (2013) Vectorized data acquisition and fast triple-correlation integrals for Fluorescence Triple Correlation Spectroscopy. Comput Phys Commun 184:1322-1332
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
Puglisi, Joseph D; Williamson, James R (2010) Nucleic acids continue to surprise. Curr Opin Struct Biol 20:259-61
Bunner, Anne E; Williamson, James R (2009) Stable isotope pulse-chase monitored by quantitative mass spectrometry applied to E. coli 30S ribosome assembly kinetics. Methods 49:136-41
Sykes, Michael T; Williamson, James R (2009) A complex assembly landscape for the 30S ribosomal subunit. Annu Rev Biophys 38:197-215

Showing the most recent 10 out of 35 publications