The [4Fe‐4S] clusters of Rpo3 are key determinants in the post Rpo3/Rpo11 heterodimer formation of RNA polymerase in Methanosarcina acetivorans. Issue 1 (25th August 2016)

• Record Type: Journal Article
• Authors: Jennings, Matthew E.; Lessner, Faith H.; Karr, Elizabeth A.; Lessner, Daniel J
• Is Part Of: MicrobiologyOpen. Volume 6:Issue 1(2017:Feb.)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2016-08-25
• Subjects: anaerobes -- archaea -- iron–sulfur cluster -- transcription; Microbiology -- Periodicals; 579
• DOI: 10.1002/mbo3.399
• Languages: English
• ISSNs: 2045-8827

Abstract

Abstract: Subunits Rpo3 and Rpb3/AC40 of RNA polymerase (RNAP) from many archaea and some eukaryotes, respectively, contain a ferredoxin‐like domain (FLD) predicted to bind one or two [4Fe‐4S] clusters postulated to play a role in regulating the assembly of RNAP. To test this hypothesis, the two [4Fe‐4S] cluster Rpo3 from Methanosarcina acetivorans was modified to generate variants that lack the FLD or each [4Fe‐4S] cluster. Viability of gene replacement mutants revealed that neither the FLD nor the ability of the FLD to bind either [4Fe‐4S] cluster is essential. Nevertheless, each mutant demonstrated impaired growth due to significantly lower RNAP activity when compared to wild type. Affinity purification of tagged Rpo3 variants from M. acetivorans strains revealed that neither the FLD nor each [4Fe‐4S] cluster is required for the formation of a Rpo3/11 heterodimer, the first step in the assembly of RNAP. However, the association of the Rpo3/11 heterodimer with catalytic subunits Rpo2′ and Rpo1″ was diminished by the removal of the FLD and each cluster, with the loss of cluster 1 having a more substantial effect than the loss of cluster 2. These results reveal that the FLD and [4Fe‐4S] clusters, particularly cluster 1, are key determinants in the post Rpo3/11 heterodimer assembly of RNAP in M. acetivorans . Abstract : The Rpo3 subunit of RNA polymerase (RNAP) from numerous species of archaea contains a ferredoxin‐like domain harboring [4Fe‐4S] cluster(s), the function of which is unknown. The two [4Fe‐4S] cluster Rpo3 from Methanosarcina acetivorans was mutated and characterized, which revealed that the clusters are not essential, but are important factors in the formation of RNAP after the assembly of the Rpo3/Rpo11 heterodimer.