Protein synthesis is an essential cellular process, and translation by the ribosome has been an area of active research for many decades. Recently, new techniques such as single-molecule fluorescence resonance energy transfer and a wealth of structural information have yielded tremendous insight into the mechanism of ribosome-catalyzed protein synthesis. However, imaging translating ribosomes in living cells has been hindered by technical limitations that include poor brightness and photostability of genetically-encoded labels, poor labeling of cellular ribosomes, and low signal-to-noise during experimental measurement. In addition, recently developed technologies to monitor translation in living cells such as ribosome profiling, while powerful, are time-consuming, expensive, and monitor an ensemble of ribosomes rather than single ribosomes. The proposed research is designed to overcome these limitations in order to study the mechanism of translation at the single-molecule level in living cells. This research employs a novel RNA mimic of GFP called Spinach, which is an RNA aptamer that binds a fluorophore to become fluorescent. Spinach is advantageous over currently available labels because it is genetically-encoded, bright, and photostable. In addition, it is ideally suited for labeling ribosomes because its sequence can be directly inserted into ribosomal RNA at tolerated positions. Imaging active ribosomes in cells will represent a huge step forward in the study of translation, and will enable numerous experiments regarding the basic mechanism and regulation of translation in a cellular context, thus allowing for crucial new insights into how factors such as antibiotics, translation factors, and cellular stresses affect translation. Indeed, one aim of the proposed research is to clarify the biological role of the release factor RF3 during translation. Moreover, the proposed research can be directly expanded to label and image eukaryotic and ribosomes and begin addressing important questions regarding the mechanism and regulation of eukaryotic translation in numerous systems. This includes cells such as neurons, which are known to have highly regulated local translation that is crucial to cell growth and function.
Protein translation is a fundamental process in molecular biology that is crucial for health and viability of all life. The proposed research serves to study the mechanism of protein translation by developing a novel approach to image actively translating ribosomes at the single-molecule level in living bacteria. The proposed work will represent a major breakthrough in the study of translation by yielding unprecedented views of this dynamic process in real time in living cells.
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