Related Experiment Video
Updated: Mar 31, 2026

Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli pppGpp Followed by Thin Layer Chromatography
Published on: June 4, 2019
Effects of amino acid starvation on RelA diffusive behavior in live Escherichia coli
Wenting Li1, Emmanuelle Bouveret2, Yan Zhang3
1Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA.
Abstract:
During amino acid starvation, bacterial cells rapidly synthesize the nucleotides (p)ppGpp, causing a massive re-programming of the transcriptional profile known as the stringent response. The (p)ppGpp synthase RelA is activated by ribosomes harboring an uncharged tRNA at the A site. It is unclear whether synthesis occurs while RelA is bound to the ribosome or free in the cytoplasm. We present a study of three Escherichia coli strains, each expressing a different RelA-fluorescent protein (RelA-FP) construct: RelA-YFP, RelA-mEos2 and RelA-Dendra2. Single-molecule localization and tracking studies were carried out under normal growth conditions and during amino acid starvation. Study of three labeling schemes enabled us to assess potential problems with FP labeling of RelA. The diffusive trajectories and axial spatial distributions indicate that amino acid starvation induces net binding of all three RelA-FP constructs to 70S ribosomes. The data are most consistent with a model in which RelA synthesizes (p)ppGpp while bound to the 70S ribosome. We suggest a 'short hopping time' model of RelA activity during starvation. Our results contradict an earlier study of RelA-Dendra2 diffusion that inferred off-ribosome synthesis of (p)ppGpp. The reasons for the discrepancy remain unclear.
More Related Videos
10:34Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli
Published on: August 22, 2017
09:49Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection
Published on: November 18, 2022
Related Concept Videos
Stringent Response in E. coli
Protein Diffusion in the Membrane
Chemotaxis in E. coli
Amino Acid Catabolism
Protein Dynamics in Living Cells
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
Evolution of New Traits in Microbes