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Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu

Transcription and translation of mRNA's are coordinated processes in bacteria. We have previously shown that a mutant form of EF-Tu (Gln125Arg) in Salmonella Typhimurium with a reduced affinity for aa-tRNA, causes ribosome pausing, resulting in an increased rate of RNase E-mediated mRNA cleavag...

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Autores principales: Bergman, Jessica M., Hammarlöf, Disa L., Hughes, Diarmaid
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3938759/
https://www.ncbi.nlm.nih.gov/pubmed/24587376
http://dx.doi.org/10.1371/journal.pone.0090486
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author Bergman, Jessica M.
Hammarlöf, Disa L.
Hughes, Diarmaid
author_facet Bergman, Jessica M.
Hammarlöf, Disa L.
Hughes, Diarmaid
author_sort Bergman, Jessica M.
collection PubMed
description Transcription and translation of mRNA's are coordinated processes in bacteria. We have previously shown that a mutant form of EF-Tu (Gln125Arg) in Salmonella Typhimurium with a reduced affinity for aa-tRNA, causes ribosome pausing, resulting in an increased rate of RNase E-mediated mRNA cleavage, causing extremely slow growth, even on rich medium. The slow growth phenotype is reversed by mutations that reduce RNase E activity. Here we asked whether the slow growth phenotype could be reversed by overexpression of a wild-type gene. We identified spoT (encoding ppGpp synthetase/hydrolase) as a gene that partially reversed the slow growth rate when overexpressed. We found that the slow-growing mutant had an abnormally high basal level of ppGpp that was reduced when spoT was overexpressed. Inactivating relA (encoding the ribosome-associated ppGpp synthetase) also reduced ppGpp levels and significantly increased growth rate. Because RelA responds specifically to deacylated tRNA in the ribosomal A-site this suggested that the tuf mutant had an increased level of deacylated tRNA relative to the wild-type. To test this hypothesis we measured the relative acylation levels of 4 families of tRNAs and found that proline isoacceptors were acylated at a lower level in the mutant strain relative to the wild-type. In addition, the level of the proS tRNA synthetase mRNA was significantly lower in the mutant strain. We suggest that an increased level of deacylated tRNA in the mutant strain stimulates RelA-mediated ppGpp production, causing changes in transcription pattern that are inappropriate for rich media conditions, and contributing to slow growth rate. Reducing ppGpp levels, by altering the activity of either SpoT or RelA, removes one cause of the slow growth and reveals the interconnectedness of intracellular regulatory mechanisms.
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spelling pubmed-39387592014-03-04 Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu Bergman, Jessica M. Hammarlöf, Disa L. Hughes, Diarmaid PLoS One Research Article Transcription and translation of mRNA's are coordinated processes in bacteria. We have previously shown that a mutant form of EF-Tu (Gln125Arg) in Salmonella Typhimurium with a reduced affinity for aa-tRNA, causes ribosome pausing, resulting in an increased rate of RNase E-mediated mRNA cleavage, causing extremely slow growth, even on rich medium. The slow growth phenotype is reversed by mutations that reduce RNase E activity. Here we asked whether the slow growth phenotype could be reversed by overexpression of a wild-type gene. We identified spoT (encoding ppGpp synthetase/hydrolase) as a gene that partially reversed the slow growth rate when overexpressed. We found that the slow-growing mutant had an abnormally high basal level of ppGpp that was reduced when spoT was overexpressed. Inactivating relA (encoding the ribosome-associated ppGpp synthetase) also reduced ppGpp levels and significantly increased growth rate. Because RelA responds specifically to deacylated tRNA in the ribosomal A-site this suggested that the tuf mutant had an increased level of deacylated tRNA relative to the wild-type. To test this hypothesis we measured the relative acylation levels of 4 families of tRNAs and found that proline isoacceptors were acylated at a lower level in the mutant strain relative to the wild-type. In addition, the level of the proS tRNA synthetase mRNA was significantly lower in the mutant strain. We suggest that an increased level of deacylated tRNA in the mutant strain stimulates RelA-mediated ppGpp production, causing changes in transcription pattern that are inappropriate for rich media conditions, and contributing to slow growth rate. Reducing ppGpp levels, by altering the activity of either SpoT or RelA, removes one cause of the slow growth and reveals the interconnectedness of intracellular regulatory mechanisms. Public Library of Science 2014-02-28 /pmc/articles/PMC3938759/ /pubmed/24587376 http://dx.doi.org/10.1371/journal.pone.0090486 Text en © 2014 Bergman et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Bergman, Jessica M.
Hammarlöf, Disa L.
Hughes, Diarmaid
Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu
title Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu
title_full Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu
title_fullStr Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu
title_full_unstemmed Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu
title_short Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu
title_sort reducing ppgpp level rescues an extreme growth defect caused by mutant ef-tu
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3938759/
https://www.ncbi.nlm.nih.gov/pubmed/24587376
http://dx.doi.org/10.1371/journal.pone.0090486
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