The [4Fe‐4S] clusters of Rpo3 are key determinants in the post Rpo3/Rpo11 heterodimer formation of RNA polymerase in Methanosarcina acetivorans

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.

Methanosarcina acetivorans contains a functional ISC system for iron-sulfur cluster biogenesis

“…M. acetivorans strain WWM73 was obtained from Dr. Bill Metcalf [ 52 ] and was used as the parent strain for all experiments. All strains of M. acetivorans (Table S 1 ) were grown in HS medium containing 125 mM methanol as a carbon and energy source as previously described [ 53 , 54 ]. Each liter of HS medium contains 23.4 g NaCl, 3.8 g NaHCO 3 , 1.0 g KCl, 11.0 g MgCl 2 *6H 2 O, 0.3 g CaCl 2 *2H 2 O, 1.0 g NH 4 Cl, 0.5 g L-cysteine, 5 mL of 1 M KH 2 PO 4 at pH = 7.4, 1 mL of 0.1% w/v resazurin, 10 mL of Wolfe’s Mineral Solution (supplemented with 0.024 g/L NiCl 2 *H 2 O in the stock), and 2 mL of a 5x concentrated Wolfe’s Vitamin Solution [ 55 ].…”

“…acetivorans strain WWM73 was obtained from Dr. Bill Metcalf [52] and was used as the parent strain for all experiments. All strains of M. acetivorans (Table S1) were grown in HS medium containing 125 mM methanol as a carbon and energy source as previously described [53,54]. Each liter of HS medium contains 23.4 g NaCl, 3. sterile, anoxic stock just prior to inoculation.…”

Redox Chemistry in the Genome: Emergence of the [4Fe4S] Cofactor in Repair and Replication

“…Finally RNA polymerase II, which synthesizes RNA during transcription, has been demonstrated to contain a [4Fe4S] cluster. (29, 112, 141). These proteins may use redox signaling on DNA to coordinate activity in cells, and investigation of their [4Fe4S] redox properties will illuminate important biochemical features of the proteins and their pathways.…”