title: Single-molecule investigations of the stringent response machinery in living bacterial cells.
abstract: 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.
authors: Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.
authors: Brian P
authors: BP
authors: English
authors: Vasili
authors: V
authors: Hauryliuk
authors: Arash
authors: A
authors: Sanamrad
authors: Stoyan
authors: S
authors: Tankov
authors: Nynke H
authors: NH
authors: Dekker
authors: Johan
authors: J
authors: Elf
mesh_terms: D000596:Amino Acids / Q000494:pharmacology; D055162:Biocatalysis; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D003198:Computer Simulation; D003600:Cytosol / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D006159:Guanosine Tetraphosphate / Q000378:metabolism; D007700:Kinetics; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008715:Methionine / Q000494:pharmacology; D018613:Microscopy, Confocal; D012270:Ribosomes / Q000378:metabolism*; D013997:Time Factors; D014364:Tryptophan / Q000494:pharmacology
chemical_list: D000596:Amino Acids; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D006159:Guanosine Tetraphosphate; D014364:Tryptophan; D008715:Methionine; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases