Time-Resolved Hydroxyl Radical Footprinting of RNA

Johns Hopkins University, Baltimore MD

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Abstract

The synthesis of new ribosomes is essential for cell growth, and tightly regulated in response to nutrients and stress. Defects in ribosome synthesis are linked to increased risk of cancer and a variety of congenital defects in rapidly growing tissues such as blood and skin. As the RNA and protein components of the ribosome assemble with each other to form the large and small subunits of the ribosome, local protein-RNA interactions drive programmed conformational changes in the RNA alter the direction and frequency of helix motions. These protein-guided changes in the RNA dynamics reorder the relative free energies of assembly intermediates and control the hierarchical assembly of additional proteins to the complex. The proposed research will use single molecule fluorescence and in-cell RNA structure probing to investigate how conformational switches coordinate assembly of individual domains of the 30S ribosome, and how assembly of each domain is linked to folding of the active site, which lies at their intersection. Single-molecule pull-down experiments and in vivo footprinting of nascent transcripts will visualize how assembly factors increase the fidelity and cooperativity of assembly during transcription of the pre-rRNA. The results of this research will increase understanding of how large RNA-protein complexes necessary for cell growth are assembled in the cell. It will also contribute to a molecular explanation of human disorders arising from defects in the assembly of these complexes.