Effects of amino acid starvation on RelA diffusive behavior in live Escherichia coli.
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.
Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA.
Wenting
W
Li
Laboratory of Macromolecular System Engineering (LISM), Centre National de la Recherche Scientifique (CNRS), Aix-Marseille University, Marseille, France.
Emmanuelle
E
Bouveret
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA.
Yan
Y
Zhang
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA.
Kuanqing
K
Liu
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA.
Jue D
JD
Wang
Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA.|Molecular Biophysics Program, University of Wisconsin-Madison, Madison, WI, USA.
James C
JC
Weisshaar
D000596:Amino Acids / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases