文章基本信息

标题：Bordetella pertussis fim3 gene regulation by BvgA: Phosphorylation controls the formation of inactive vs. active transcription complexes
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作者：Alice Boulanger ; Kyung Moon ; Kimberly B. Decker 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：6
页码：E526-E535
DOI：10.1073/pnas.1421045112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceThe response regulator BvgA controls virulence gene expression in the human pathogen Bordetella pertussis. Phosphorylated BvgA together with RNA polymerase form transcription complexes that synthesize RNA from the B. pertussis promoter for the fimbrial gene fim3. We show that nonphosphorylated BvgA and RNA polymerase form stable but inactive complexes. We propose that these complexes may modulate fim3 expression under inducing conditions and facilitate rapid repression of the fim3 promoter under noninducing conditions. The ability of a response regulator to activate and repress depending on environmental conditions represents a different paradigm in two-component system regulation. Two-component systems [sensor kinase/response regulator (RR)] are major tools used by microorganisms to adapt to environmental conditions. RR phosphorylation is typically required for gene activation, but few studies have addressed how and if phosphorylation affects specific steps during transcription initiation. We characterized transcription complexes made with RNA polymerase and the Bordetella pertussis RR, BvgA, in its nonphosphorylated or phosphorylated (BvgA[~]P) state at Pfim3, the promoter for the virulence gene fim3 (fimbrial subunit), using gel retardation, potassium permanganate and DNase I footprinting, cleavage reactions with protein conjugated with iron bromoacetamidobenzyl-EDTA, and in vitro transcription. Previous work has shown that the level of nonphosphorylated BvgA remains high in vivo under conditions in which BvgA is phosphorylated. Our results here indicate that surprisingly both BvgA and BvgA[~]P form open and initiating complexes with RNA polymerase at Pfim3. However, phosphorylation of BvgA is needed to generate the correct conformation that can transition to competent elongation. Footprints obtained with the complexes made with nonphosphorylated BvgA are atypical; while the initiating complex with BvgA synthesizes short RNA, it does not generate full-length transcripts. Extended incubation of the BvgA/RNA polymerase initiated complex in the presence of heparin generates a stable, but defective species that depends on the initial transcribed sequence of fim3. We suggest that the presence of nonphosphorylated BvgA down-regulates Pfim3 activity when phosphorylated BvgA is present and may allow the bacterium to quickly adapt to the loss of inducing conditions by rapidly eliminating Pfim3 activation once the signal for BvgA phosphorylation is removed.
关键词：transcription ; BvgA ; RNA polymerase ; Bordetella pertussis ; fim3