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- W2023376768 abstract "We have developed a single-molecule fluorescence assay to directly observe the stringent response in individual living E. coli cells. For this purpose, we have created chromosomal fusions of both RelA and three ribosomal proteins with a photo-activatable fluorescent protein.The stringent factor RelA binds to a small fraction of ribosomes, where it synthesizes the global transcriptional regulator ppGpp in response to amino acids deprivation. Our objective is to study the binding kinetics of individual RelA molecules to the ribosome in living cells and to observe how its kinetics changes during a nutritional down-shift.While E. coli contains on average about 100 RelA molecules and 20000 ribosomes, using a photo-activatable fluorescent probe we can activate only a few fluorescent molecules per cell at any given time. We induce stringent response by rapid addition of amino acid hydroxamates. Since our fluorescent tag is photoconvertible, we can repeat tracking experiments many times in the same E. coli cell.We record trajectories of individual RelA molecules diffusing in living E. coli cells with a laser exposure of 1 millisecond, a frame time of 5 milliseconds, and a spatial precision of 50 nanometers. The high resolution of the experiments makes it possible to characterize RelA binding kinetics under varying growth conditions.When the cell grows exponentially, RelA trajectories closely resemble trajectories of fluorescently tagged ribosomal proteins. After nutritional downshift, RelA binding kinetics changes rapidly. Results suggest that under amino-acid starvation, RelA is only transiently bound to the ribosome. The data is consistent with an order of magnitude drop in affinity to the ribosome." @default.
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- W2023376768 date "2010-01-01" @default.
- W2023376768 modified "2023-09-28" @default.
- W2023376768 title "Single Molecule Tracking Inside Individual Living Bacterial Cells" @default.
- W2023376768 doi "https://doi.org/10.1016/j.bpj.2009.12.3190" @default.
- W2023376768 hasPublicationYear "2010" @default.
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