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Subunit D of RNA Polymerase from Methanosarcina acetivorans Contains Two Oxygen-labile [4Fe-4S] Clusters IMPLICATIONS FOR OXIDANT-DEPENDENT REGULATION OF TRANSCRIPTION

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dc.contributorEduardus C. Duin, duinedu@auburn.eduen_US
dc.creatorLessner, Faith H.
dc.creatorJennings, Matthew E.
dc.creatorHirata, Akira
dc.creatorDuin, Eduardus C.
dc.creatorLessner, Daniel J.
dc.description.abstractSubunit D of multisubunit RNA polymerase from many species of archaea is predicted to bind one to two iron-sulfur (Fe-S) clusters, the function of which is unknown. A survey of encoded subunit D in the genomes of sequenced archaea revealed six distinct groups based on the number of complete or partial [4Fe-4S] cluster motifs within domain 3. Only subunit D from strictly anaerobic archaea, including all members of the Methanosarcinales, are predicted to bind two [4Fe-4S] clusters. We report herein the purification and characterization of Methanosarcina acetivorans subunit D in complex with subunit L. Expression of subunit D and subunit L in Escherichia coli resulted in the purification of a D-L heterodimer with only partial [4Fe-4S] cluster content. Reconstitution in vitro with iron and sulfide revealed that the M. acetivorans D-L heterodimer is capable of binding two redox-active [4Fe-4S] clusters. M. acetivorans subunit D deleted of domain 3 (D Delta D3) was still capable of co-purifying with subunit L but was devoid of [4Fe-4S] clusters. Affinity purification of subunit D or subunit D Delta D3 from M. acetivorans resulted in the co-purification of endogenous subunit L with each tagged subunit D. Overall, these results suggest that domain 3 of subunit D is required for [4Fe-4S] cluster binding, but the [4Fe-4S] clusters and domain 3 are not required for the formation of the D-L heterodimer. However, exposure of two [4Fe-4S] cluster-containing D-L heterodimer to oxygen resulted in loss of the [4Fe-4S] clusters and subsequent protein aggregation, indicating that the [4Fe-4S] clusters influence the stability of the D-L heterodimer and therefore have the potential to regulate the assembly and/or activity of RNA polymerase in an oxidant-dependent manner.en_US
dc.publisherAmerican Society for Biochemistry and Molecular Biologyen_US
dc.relation.ispartofJournal of Biological Chemistryen_US
dc.subjectArchaea Iron-sulfur protein Oxidative stress Redox regulation RNA polymerase Transcription methanogenen_US
dc.titleSubunit D of RNA Polymerase from Methanosarcina acetivorans Contains Two Oxygen-labile [4Fe-4S] Clusters IMPLICATIONS FOR OXIDANT-DEPENDENT REGULATION OF TRANSCRIPTIONen_US
dc.type.genreJournal Article, Academic Journalen_US

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