A Fraction of the BglG Transcriptional Antiterminator from Escherichia coli Exists as a Compact Monomer

Fux, Liat; Nussbaum-Shochat, Anat; Amster‐Choder, Orna

doi:10.1074/jbc.m308085200

Cited by 9 publications

(19 citation statements)

References 30 publications

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“…The requirement for the conserved histidines in PRD1 for the EII-catalyzed phosphorylation on PRD2, observed in the case of BglG and other antiterminators, might reflect the importance of these residues for BglG folding into a phosphorylatable conformation. Indeed, PRD1 and PRD2 of BglG could be cross-linked in vitro and in vivo, indicating that a fraction of BglG folds into a compact conformation in which these domains are in a very close proximity (22).…”

Section: Discussionmentioning

confidence: 99%

“…We attempted to reproduce transcription antitermination in this minimal sys-tem by adding purified and soluble BglG fused to maltosebinding protein (MBP). MBP-BglG was shown to be active in bgl antitermination in vivo and to be regulated by BglF in vitro and in vivo (20,22). As a template, we used a 532-bp PCR fragment containing the bgl promoter activated by a point mutation in the CAP-binding site (23), the leader region, which contains a transcription terminator, and the very beginning of the bglG gene (encoding the first 19 aa of BglG).…”

Section: Resultsmentioning

confidence: 99%

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Modulation of transcription antitermination in the bgl operon of Escherichia coli by the PTS

Raveh¹,

Lopian²,

Nussbaum-Shochat³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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BglG, which regulates expression of the ␤-glucoside utilization (bgl) operon in Escherichia coli, represents a family of RNA-binding transcriptional antiterminators that positively regulate transcription of sugar utilization genes in Gram-negative and Gram-positive organisms. BglG is negatively regulated by the ␤-glucoside phosphotransferase, BglF, by means of phosphorylation and physical association, and it is positively regulated by the general phosphoenolpyruvate phosphotransferase system (PTS) proteins, enzyme I (EI) and HPr. We studied the positive regulation of BglG both in vitro and in vivo. Here, we show that although EI and HPr are essential for BglG activity, this mode of activation does not require phosphorylation of BglG by HPr, as opposed to the phosphorylation-mediated activation of many BglG-like antiterminators in Gram-positive organisms. The effect of EI and HPr on BglG is not mediated by BglF. Nevertheless, the release of BglG from BglF, which is stimulated by the extracellular sugar in a sugar uptakeindependent manner, is a prerequisite for BglG activation. Taken together, the results indicate that activation of BglG is a 2-stage process: a sugar-stimulated release from the membrane-bound sugar sensor followed by a phosphorylation-independent stimulatory effect exerted by the general PTS proteins.bgl system ͉ E. coli

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Section: Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Modulation of transcription antitermination in the bgl operon of Escherichia coli by the PTS

Raveh¹,

Lopian²,

Nussbaum-Shochat³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…When specified, 0.5% arbutin (hydroxyphenyl-␤-D-glucopyranoside) was added to the medium. When cells reached an optical density at 600 nm of 0.6, in vivo cross-linking with p-PDM was performed as described before (12). Cells were subsequently handled and analyzed as previously described (21), except that when cells were grown in the presence of arbutin, 0.5% arbutin was maintained in all solutions throughout the procedure.…”

Section: Strainsmentioning

confidence: 99%

“…MBP-tagged BglG, PRD1, or PRD2 was expressed in MC1061 and purified as described previously for MBP-BglG (12). His-tagged enzyme I (EI), HPr, and IIB bgl were expressed in BL21(DE3).…”

Section: Strainsmentioning

confidence: 99%

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Modulation of Monomer Conformation of the BglG Transcriptional Antiterminator from Escherichia coli

et al. 2004

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The BglG protein positively regulates expression of the bgl operon in Escherichia coli by binding as a dimer to the bgl transcript and preventing premature termination of transcription in the presence of ␤-glucosides. BglG activity is negatively controlled by BglF, the ␤-glucoside phosphotransferase, which reversibly phosphorylates BglG according to ␤-glucoside availability, thus modulating its dimeric state. BglG consists of an RNAbinding domain and two homologous domains, PRD1 and PRD2. Based on structural studies of a BglG homologue, the two PRDs fold similarly, and the interactions within the dimer are PRD1-PRD1 and PRD2-PRD2. We have recently shown that the affinity between PRD1 and PRD2 of BglG is high, and a fraction of the BglG monomers folds in the cell into a compact conformation, in which PRD1 and PRD2 are in close proximity. We show here that both BglG forms, the compact and noncompact, bind to the active site-containing domain of BglF, IIB bgl , in vitro. The interaction of BglG with IIB bgl or BglF is mediated by PRD2. Both BglG forms are detected as phosphorylated proteins after in vitro phosphorylation with IIB bgl and are dephosphorylated by BglF in vitro in the presence of ␤-glucosides. Nevertheless, genetic evidence indicates that the interaction of IIB bgl and BglF with the compact form is seemingly less favorable. Using in vivo cross-linking, we show that BglF enhances folding of BglG into a compact conformation, whereas the addition of ␤-glucosides reduces the amount of this form. Based on these results we suggest a model for the modulation of BglG conformation and activity by BglF.The expression of the bgl operon in Escherichia coli, whose products are required for ␤-glucoside utilization, is regulated by two of its gene products, BglG, a transcriptional regulator, and BglF, a membrane-bound sugar sensor (4). Transcription from the bgl promoter initiates constitutively but, in the absence of ␤-glucosides, most transcripts terminate prematurely at one of the two -independent terminators that flank the first gene of the operon. In the presence of ␤-glucosides, BglG prevents termination of transcription at these sites (20, 26) by binding to specific sites on the bgl transcript, which partially overlap with each of the terminators, and stabilizing an alternative secondary structure of the RNA that enables RNA polymerase to proceed (15). BglG activity is regulated by BglF, an enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), which reversibly phosphorylates BglG according to ␤-glucoside availability (1, 3, 25), thus modulating its dimeric state (2). Hence, in the absence of ␤-glucosides, BglG exists as an inactive, phosphorylated monomer, whereas in the presence of ␤-glucosides in the growth medium, BglG exists as an active nonphosphorylated dimer (2). A recent publication from our laboratory demonstrated that BglF recruits BglG to form a precomplex at the cell membrane and releases it to the cytoplasm upon the addition of ␤-glucosides (17).BglG represent a family o...

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A Quantitative bgl Operon Model for E. coli Requires BglF Conformational Change for Sugar Transport

Chopra

Bender

2008

Lecture Notes in Computer Science

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A Fraction of the BglG Transcriptional Antiterminator from Escherichia coli Exists as a Compact Monomer

Cited by 9 publications

References 30 publications

Modulation of transcription antitermination in the bgl operon of Escherichia coli by the PTS

Modulation of transcription antitermination in the bgl operon of Escherichia coli by the PTS

Modulation of Monomer Conformation of the BglG Transcriptional Antiterminator from Escherichia coli

A Quantitative bgl Operon Model for E. coli Requires BglF Conformational Change for Sugar Transport

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