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Single-Cell Biophysics: Measurement, Modulation, and Modeling
Poster Abstracts
111
26-POS
Board 13
Tracking Orthogonal Fluorescent Ribosomes inside Living Cells
Arvid H. Gynnå
, Ivan L. Volkov, Johan Elf.Magnus Johansson.
Uppsala University, Uppsala, Sweden.
The kinetic parameters of protein synthesis have been successfully studied by in vitro methods
for decades. There are however many differences between the cellular environment and
reconstituted systems that might affect these parameters. Among those are molecular crowding,
confinement, uncertain intracellular ionic strength and pH, and possibly even the presence of yet
unknown factors. Current methods for in vivo investigation of protein synthesis are limited. They
are normally indirect, and/or give average numbers for several different molecular species. A
method to directly measure the translation rate of individual ribosomes performing well-defined
functions inside living cells is hence needed. To define the function of the ribosomes under
observation inside a cell, a subset of the ribosomes should preferentially be orthogonal so they
will only translate the mRNA of interest. In an organism with several rRNA loci, this can be
achieved by modifying the anti-Shine-Dalgarno sequence of one of them and expressing a
correspondingly orthogonal mRNA. For use in single-particle tracking experiments, the
orthogonal ribosome subset must also be fluorescently labeled. We have developed such a
system for
E. coli
, where the orthogonal small ribosomal subunits are labeled with an organic
dye and thus can be monitored in singulo while operating inside the cell interacting with all
natural components. This opens up the possibility to measure in vivo translation rates on defined
mRNAs, as well as mapping the physical locations of the synthesis of different proteins.