title: Subunit D of RNA polymerase from Methanosarcina acetivorans contains two oxygen-labile [4Fe-4S] clusters: implications for oxidant-dependent regulation of transcription.
abstract: Subunit 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 (DΔD3) was still capable of co-purifying with subunit L but was devoid of [4Fe-4S] clusters. Affinity purification of subunit D or subunit DΔD3 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.
authors.affiliation: Department of Biological Sciences, University of Arkansas, Fayetteville, Arkansas 72701, USA.
authors.forename: Faith H
authors.initials: FH
authors.lastname: Lessner
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Jennings
authors.forename: Akira
authors.initials: A
authors.lastname: Hirata
authors.forename: Eduardus C
authors.initials: EC
authors.lastname: Duin
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Lessner
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D005786:Gene Expression Regulation; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D017020:Methanosarcina / Q000201:enzymology*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
chemical_list: D017931:DNA Primers; D007506:Iron-Sulfur Proteins; D012321:DNA-Directed RNA Polymerases; D010100:Oxygen