This application is for an individual fellowship for an MD-PhD student, with a research training plan designed to aid his long-term goal of becoming an independent physician scientist in the field of virology. Many viruses (including HIV, SARS, and West Nile) evolved an ability to regularly trigger frameshifts by one nucleotide~ these viral programmed ribosomal frameshifts (PRFs) produce proteins from overlapping reading frames of the same mRNA. It is unknown what structural changes occur in the ribosome during PRF, or how downstream mRNA 2? structures enhance the efficiency of PRF at 7-nucleotide slippery sequences. This application proposes using single molecule FRET (smFRET) to study dynamic, frameshifting ribosomes to distinguish between competing models of PRF mechanism. Preliminary smFRET characterization of the behavior of dynamically translating ribosomes when not undergoing PRF revealed a novel state seen only during active translation.
In aim 1, smFRET study of dynamic prokaryotic and eukaryotic ribosomes will be extended to determine if that novel conformation corresponds to global structural changes during multiple substeps of translation.
In aim 2, smFRET of dynamic ribosomes on viral mRNA will be used to determine the mechanism of PRF, namely the structural and kinetic properties unique to ribosomes that frameshift, how mRNA 2? structures enhance PRF efficiency, and mechanistic similarities and differences of PRF in multiple viruses (IBV, SARS, HIV). These experimental results will distinguish between competing models of PRF, giving a detailed understanding of this process central to viral pathogenesis.
Viral hijacking of host cell translational machinery is amongst the most central, but least understood mechanisms essential to pathogens such as HIV, SARS, and West Nile. This application proposes the study of dynamic, frameshifting ribosomes at a single-molecule resolution to distinguish between competing models of viral programmed ribosomal frameshifting (PRF) mechanism. Disruption of viral PRF can hinder viral replication, thus a detailed understanding could ultimately enable therapeutic targeting of that process.
| Jamiolkowski, Ryan M; Chen, Chunlai; Cooperman, Barry S et al. (2017) tRNA Fluctuations Observed on Stalled Ribosomes Are Suppressed during Ongoing Protein Synthesis. Biophys J 113:2326-2335 |
