Synthesis of proteins via the translation of messenger RNAs (mRNAs) by the cellular translation machinery (TM) is a fundamental step in gene expression that is central to life. Because the bacterial TM is a proven target for the development of new antibiotics and because a growing list of human diseases have been causally linked to aberrant control of translation, the bacterial and eukaryotic TM remain under intense investigation. Over the past 15 years, structural studies have highlighted the large-scale structural rearrangements that the TM must seemingly undergo during protein synthesis. Unfortunately, the size, complexity, and inherent conformational flexibility of the TM have made it challenging to characterize these dynamics, greatly limiting our understanding of how they contribute to the mechanism and control of translation. Nonetheless, we and others have recently developed experimental methods that have enabled us to use single-molecule fluorescence (SMF) approaches to characterize the dynamics of the bacterial TM during conventional protein synthesis. Despite these efforts, a critical gap in our understanding remains regarding how the dynamics of the conventional process are altered and/or manipulated to drive unconventional, but biomedically important, processes involved in translational control. In the first aim of this proposal, we begin to address this gap by using a combination of molecular biological-, SMF-, biophysical-, biochemical-, and structural approaches to investigate how the dynamics of the bacterial TM are manipulated in order to drive a wide-spread translational control strategy known as frameshifting. During frameshifting, the TM either skips or re-reads one or two nucleotides within an mRNA to either correct an insertion or deletion mutation that would otherwise result in production of an aberrant or truncated protein or to drive the alternative expression of the mRNA to produce a specific protein. These experiments promise to reveal the still-elusive mechanism(s) that underlie frameshifting. In the second aim, we will use analogous approaches to investigate how regulatory translation factors and antibiotics manipulate the dynamics of the bacterial TM in order to suppress or induce frameshifting. These studies will elucidate strategies that nature uses to control frameshifting and which we hope will inform the development of new antibiotics and other therapeutics that function by manipulating frameshifting. Finally, in the third aim, we will use an experimental platform that we have now established for SMF studies of translation in yeast to characterize the dynamics of the eukaryotic TM, that the mechanisms through which translation factor-mediated regulation of these dynamics drive eukaryotic translation, and the effects that eukaryote-specific post-translational modifications of translation factors have on the ability of these factors to regulate the dynamics of the eukaryotic TM and drive eukaryotic translation. These studies will enable a highly detailed understanding of the mechanisms that drive and regulate eukaryotic translation and hold tremendous promise for elucidating the role of translational control in human health and disease.
As has been widely and recently reported by the National Institutes of Health, the World Health Organization, and newspapers such as the New York Times, the world is currently facing a rapidly emerging antibacterial antibiotic resistance crisis, and the ribosome, which is the universally distributed biological machine that is ultimately responsible for synthesizing all of te proteins encoded in an organism's DNA genome, serves as the molecular target of over half of the antibacterial antibiotics that are in current, clinical use. In addition, aberrant cellular conrol over the process through which the human ribosome synthesizes proteins plays a causal role in several human diseases, including numerous viral infections and cancers. This proposal aims to elucidate the process through which the ribosome synthesizes proteins and how that process is controlled, in greater detail than is currently available, thereby aiding the development of next-generation antibacterial antibiotics as well as other small-molecule drugs that can be used to treat viral infections, cancers, and other human diseases.
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