Recent advances in structural biology have enormously changed our understanding of how proteins are synthesized, how several antibiotics selectively mediate their effects and how to predict potential new antibiotic targets. They have also provided a framework for new genetic and physical investigations to further elucidate the functional significance of ribosome conformational changes. Not all protein synthesis involves invariant successive triplet decoding or the standard meaning of codons. One of the most dramatic departures is a case of receding where half the translating ribosomes bypass a 50 nucleotide sequence present in mature mRNA. The work proposed here is designed to capitalize on the unique aspects of this system to better understand the flexibility of the protein synthetic apparatus.
Aims will involve mutational analyses of the mRNA signals responsible for bypassing and characterization of their effects by mass spectrometric analyses of the protein products (Aim 1), collaborative functional analyses by cryo-electron microscopy and fluorescence spectroscopy using stopped-flow and single molecule techniques (Aim 2) and selection and characterization of cellular mutants to reveal crucial components of bypassing (Aim 3).
Aim 4 is to maintain and improve a database of all receding events (locally reprogrammed readout) and to exploit it to help correct gene annotation and extend our knowledge about receding. Clinically important examples of receding contained in the database range from, frameshifting to sense human cellular polyamine levels as part of an autpregulatory circuit, decoding SARS-CoV, HIV RNA and human PEG10, to the essential specification of selenocysteine by certain UGA codons. The study of bypassing signals and their interaction with translating ribosomes will provide insights into both standard decoding and the expanded potential of receding. Our long range goal is to understand the extent, functions and mechanisms of receding, to manipulate the efficiency of receding in order to disrupt viral propagation and abrogate certain disease states. A related goal is to create new receding signals in trans to counteract frameshift mutations and premature stop codons implicated in human diseases.
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