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dc.contributor.authorKim, Jee-Hyun
dc.contributor.authorWei, Jun-Rong
dc.contributor.authorWallach, Joshua B.
dc.contributor.authorRobbins, Rebekkah S.
dc.contributor.authorRubin, Eric J.
dc.contributor.authorSchnappinger, Dirk
dc.date.accessioned2019-10-15T18:22:30Z
dc.date.issued2011
dc.identifier.citationKim, Jee-Hyun, Jun-Rong Wei, Joshua B. Wallach, Rebekkah S. Robbins, Eric J. Rubin, and Dirk Schnappinger. 2010. “Protein Inactivation in Mycobacteria by Controlled Proteolysis and Its Application to Deplete the Beta Subunit of RNA Polymerase.” Nucleic Acids Research 39 (6): 2210–20. https://doi.org/10.1093/nar/gkq1149.
dc.identifier.issn0305-1048
dc.identifier.issn1362-4962
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:41552056*
dc.description.abstractUsing a component of the Escherichia coli protein degradation machinery, we have established a system to regulate protein stability in mycobacteria. A protein tag derived from the E. coli SsrA degradation signal did not affect several reporter proteins in wild-type Mycobacterium smegmatis or Mycobacterium tuberculosis. Expression of the adaptor protein SspB, which recognizes this modified tag and helps deliver tagged proteins to the protease ClpXP, strongly decreased the activities and protein levels of different reporters. This inactivation did not occur when the function of ClpX was inhibited. Using this system, we constructed a conditional M. smegmatis knockdown mutant in which addition of anhydrotetracycline (atc) caused depletion of the beta subunit of RNA polymerase, RpoB. The impact of atc on this mutant was dose-dependent. Very low amounts of atc did not prevent growth but increased sensitivity to an antibiotic that inactivates RpoB. Intermediate amounts of RpoB knockdown resulted in bacteriostasis and a more substantial depletion led to a decrease in viability by up to 99%. These studies identify SspB-mediated proteolysis as an efficient approach to conditionally inactivate essential proteins in mycobacteria. They further demonstrate that depletion of RpoB by similar to 93% is sufficient to cause death of M. smegmatis.
dc.language.isoen_US
dc.publisherOxford University Press
dash.licenseLAA
dc.titleProtein inactivation in mycobacteria by controlled proteolysis and its application to deplete the beta subunit of RNA polymerase
dc.typeJournal Article
dc.description.versionVersion of Record
dc.relation.journalNucleic Acids Research
dash.depositing.authorRubin, Eric J.::2ccf3691d766f02c048797e00c06cc44::600
dc.date.available2019-10-15T18:22:30Z
dash.workflow.comments1Science Serial ID 74624
dc.identifier.doi10.1093/nar/gkq1149
dash.source.volume39;6


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