Date

July 2013

Document Type

Dissertation

Degree Name

Ph.D.

Institution

Oregon Health & Science University

Abstract

Bacillus subtilis Spx is a global transcriptional regulator that belongs to the ArsC (arsenate reductase) protein family. It bears an N-terminal C10TSC13 redox disulfide center that is oxidized in active Spx, allowing B. subtilis to survive under oxidative stress by stimulating transcription of genes that function in detoxification and restoration of thiol homeostasis. Instead of initially binding to target promoter DNA, Spx interacts with C-terminal domain of RNA polymerase (RNAP) α subunit (αCTD) to form a complex prior to target gene recognition. Previous research showed that an αCTD/Spx complex interacts with a sequence upstream of the trxB (thioredoxin reductase) promoter region (-56 to -21), specifically two putative Spx responsive cis-acting elements at positions -44 and -33 resided within target promoter trxA (thioredoxin) and trxB DNA. To investigate whether two Spx proteins interact with RNAP to activate transcription, the composition of Spx/RNAP complex was examined in vitro and in vivo by using two Spx variants with different epitopic tags in a series of affinity chromatography analyses. The result showed that only one Spx monomer interacts with RNAP to form an active complex in vitro and in vivo. In vitro affinity interaction assay and in vitro transcription also confirmed that only one Spx monomer engages in the transcriptionally active Spx/RNAP/DNA ternary complex. The G52 residue in Spx and Y263 residue in αCTD are known to constitute part of the interface of Spx and αCTD contact. Mutational analysis of Spx found that Spx(R91A) mutation affects both Spx-dependent transcriptional activation and repession, suggestive of a function of R91 in RNAP interaction. The anti-HA chromatography analysis showed that R91 residue in Spx is required for α binding to Spx. The G52R, R91A double mutant abolished transcriptional activity of Spx in vivo as well as in vitro RNAP binding ability. The results suggests that Spx has multiple contacts with the α dimer. The affinity interaction assay and far-western blotting showed that only intact α but not αCTD interacted stably with Spx. The result suggests that the intact α subunit is required for optimal α/Spx interaction, which may involve more than one contact interface. Spx redox control involves residue R92, which is conserved in ArsC where it stabilizes the active site cysteine thiolate. Intramolecular epistasis tests showed that substitution of R92 with alanine reduced Spx activity only in Spx protein that is able to form a disulfide between C10 and C13, implicating R92 in redox control of Spx activity. The Spx response cis-acting element AGCA at position -44 is conserved and was also found in another Spx-activated gene, nfrA. In vivo and in vitro transcriptional analysis showed this AGCA motif in nfrA promoter is required for Spx-dependent transcriptional activation as that in trxB promoter. The -44 AGCA element was proposed to be the site of contact for the αCTD/oxidized Spx complex. The DNA/RNAP crosslinking analysis showed that Spx, in complex with RNAP, contacted with the trxB promoter at position -44, and repositioned σ[superscript A]. Hence, a mechanism for Spx-dependent transcriptional activation is proposed here: By interacting with RNAP and the -44 element, Spx, in its oxidized form, remodels RNAP and repositions σ[superscript A] to engage the -35 and -10 core promoter elements. Proper σ[superscript A] interaction with the core promoter, mediated by Spx that is in contact with the -44 cis-acting element is required for initiating transcription from Spx-controlled promoters.

Identifier

doi:10.6083/M4QR4V4K

Division

Div. of Environmental and Biomolecular Systems

School

School of Medicine

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