Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 108, Issue 31, Pages E365-E373Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1102255108
Keywords
cytosolic diffusion; single particle tracking; photoactivated localization microscopy; stroboscopic illumination
Categories
Funding
- European Research Council
- Swedish Foundation for Strategic Research
- Swedish Research Council
- Goran Gustafssons Stiftelse
- Knut and Alice Wallenberg Foundation
- Human Frontier Science Program cross-disciplinary fellowship
- Estonian Science Foundation
- Center of Excellence in Chemical Biology
- De Jonge Akademie
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The RelA-mediated stringent response is at the heart of bacterial adaptation to starvation and stress, playing a major role in the bacterial cell cycle and virulence. RelA integrates several environmental cues and synthesizes the alarmone ppGpp, which globally reprograms transcription, translation, and replication. We have developed and implemented novel single-molecule tracking methodology to characterize the intracellular catalytic cycle of RelA. Our single-molecule experiments show that RelA is on the ribosome under nonstarved conditions and that the individual enzyme molecule stays off the ribosome for an extended period of time after activation. This suggests that the catalytically active part of the RelA cycle is performed off, rather than on, the ribosome, and that rebinding to the ribosome is not necessary to trigger each ppGpp synthesis event. Furthermore, we find fast activation of RelA in response to heat stress followed by RelA rapidly being reset to its inactive state, which makes the system sensitive to new environmental cues and hints at an underlying excitable response mechanism.
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