Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Friday Speaker Abstracts

VISUALIZATION AND QUANTIFICATION OF TRANSLATION DYNAMICS IN LIVING CELLS AT SINGLE MOLECULE RESOLUTION Tatsuya Morisaki 1 ; Stephanie L Moon 2,3 ; Anthony Khong 2,3 ; Kenneth Lyon 1 ; Roy Parker 2,3 ; Timothy J Stasevich 1,4 ; 1 Colorado State University, Biochemistry & Molecular Biology, Fort Collins, CO, USA 2 University of Colorado, Biochemistry, Boulder, CO, USA 3 Howard Hughes Medical Institute, Boulder, CO, USA 4 Tokyo Institute of Technology, World Research Hub Initiative, Yokohama, Japan While transcription processes have been imaged with single gene resolution in living cells for the past decade, it has been a challenge to directly visualize translation processes from single mRNAs in living cells. We have developed a technique to image real-time translation at single mRNA resolution in living cells utilizing multi-epitope tags and antibody-based fluorescent probes. First, by utilizing this novel technique, we have quantified the mobility of translation sites, translation initiation rates, elongation rates, and polysome occupancies at single mRNA resolution for the first time. Second, by extending this technique to a multiplex format, we have shown that the vast majority of polysomes act independently of one another, but a small fraction of polysomes formed complexes in which two distinct mRNAs can be translated simultaneously. Third, by employing this technique, we have investigated another long-standing question in gene expression regulation - translation shutoff during stress response. While it is known that global mRNAs shut off translation and get incorporated into the granules such as stress granules (SGs) and P-bodies during stress, it has not been clear when and where mRNAs shut off translation, and how mRNAs interact with these granules due to a lack of experimental techniques with sufficient spatiotemporal resolution. To address this, we have quantified the dynamic interactions between individual mRNAs, SGs, and P-bodies, along with the translation activity from each mRNA. Interestingly, we found that translating mRNAs only interact with these granules dynamically while non-translating mRNAs can form stable associations with these granules. Also, contrary to the notion of a fluid liquid phase within SGs, we discovered a subset of mRNAs inside granules that were apparently rigidly bound such that there was little to no intragranular mobility.

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