Quantifying Single mRNA Translation Kinetics in Living Cells
- 1Institute of Genome Architecture and Function and Department of Biochemistry & Molecular Biology, Colorado State University, Fort Collins, Colorado 80523
- 2Cell Biology Unit, Institute of Innovative Research, Tokyo Institute of Technology, Midori-ku, Yokohama, Kanagawa, 226-8503, Japan
- Correspondence: tim.stasevich{at}colostate.edu; tatsuya.morisaki{at}colostate.edu
SUMMARY
One of the last hurdles to quantifying the full central dogma of molecular biology in living cells with single-molecule resolution has been the imaging of single messenger RNA (mRNA) translation. Here we describe how recent advances in protein tagging and imaging technologies are being used to precisely visualize and quantify the synthesis of nascent polypeptide chains from single mRNA in living cells. We focus on recent applications of repeat-epitope tags and describe how they enable quantification of single mRNA ribosomal densities, translation initiation and elongation rates, and translation site mobility and higher-order structure. Together with complementary live-cell assays to monitor translation using fast-maturing fluorophores and mRNA-binding protein knockoff, single-molecule studies are beginning to uncover striking and unexpected heterogeneity in gene expression at the level of translation.
