Wednesday, February 5, 2025
2/5/2025
PROJECT SUMMARY Translation of messenger RNAs (mRNAs) into proteins by the ribosome and the rest of the translation machinery (TM) is a fundamental step in gene expression that is central to life. It is perhaps not surprising, then, that the bacterial TM is a proven target for the development of new antibiotics and that many human diseases have been causally linked to dysregulation of the human TM. Consequently, the mechanisms of translation and translational control in bacteria and eukaryotes remain under intense investigation. Over the past two decades, structural studies have revealed the large-scale structural rearrangements the TM undergoes during protein synthesis. Unfortunately, the size, complexity, and conformational flexibility of the TM have greatly impeded studies of these dynamics, significantly limiting our understanding of how they contribute to the mechanisms of translation and translational control. Nonetheless, using a combination of single- molecule fluorescence and structural techniques, we and others have characterized the dynamics of the core steps of translation by the bacterial TM. Despite these accomplishments, critical gaps in our understanding remain, particularly with regard to whether and how the dynamics of these core steps are modulated as part of mechanisms of many biomedically important translational control strategies. To fill these gaps, we will use a combination of single-molecule fluorescence, structural, and biochemical approaches to investigate how the dynamics of the TM are steered to execute several fundamental translational control strategies that act during initiation and elongation stages of translation in bacteria. In the case of translation initiation, we will investigate how essential protein initiation factors (IFs) modulate the conformational dynamics of the ribosomal small subunit and use conformational switching strategies to ensure the fidelity with which a ribosomal complex correctly assembles at the start codon of the mRNA to be translated. In the case of translation elongation, we will focus on ribosomal frameshifting and ribosome rescue. Ribosomal frameshifting, whereby the TM slips backward or forward by one or more nucleotides on the mRNA, may be utilized at specific mRNA locations to correct mutations that would otherwise result in production of an aberrant protein or to drive the synthesis of multiple proteins from a single mRNA. Specialized TM components have evolved to induce frameshifting at these specific locations, and our proposed studies of these features promise to reveal the still-elusive mechanisms that underlie frameshifting. Using analogous approaches, we will also investigate how ribosome rescue factors modulate the dynamics of the TM as part of the mechanisms through which they recognize and rescue translationally compromised ribosomes. These studies will provide structure-based mechanistic models of ribosome rescue systems that can inform the development of next generation small-molecule antibiotics. Using a yeast in vitro translation system we have recently developed, we will expand these studies to include eukaryotic-specific aspects of related translational control strategies in eukaryotes.
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