Orientation of Pseudomonas aeruginosa ExsA Monomers Bound to Promoter DNA and Base-Specific Contacts with the PexoT Promoter

King, Jessica M.; Brutinel, Evan D.; Marsden, Anne E.; Schubot, Florian D.; Yahr, Timothy L.

doi:10.1128/jb.00107-12

Cited by 18 publications

(40 citation statements)

References 46 publications

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“…Previous Fe-BABE footprinting experiments showed that RH1 interacts with the upstream half of binding site 1 (Ϫ50 region) and binding site 2 (Ϫ70 region) in the P exsC , P pcrG , P exoT , and P exsD promoters (12). Since ExsA binds in a head-to-tail orientation, and RH1 interacts with the GnC sequence in binding site 1, we hypothesized that RH1 of the ExsA monomer bound to site 2 also recognizes a GnC sequence.…”

Section: Resultsmentioning

confidence: 93%

“…Footprinting data obtained with Fe-BABE (a protein-labeling reagent that can be conjugated to cysteine residues), however, indicate that RH1 of the ExsA monomer bound to site 2 interacts with the Ϫ70 to Ϫ75 region of the P exoT , P exsD , P exsC , and P pcrG promoters and that RH2 interacts with the Ϫ60 region of the P exsC and P pcrG promoters (12). An interaction between RH2 and the P exoT and P exsD promoters at binding site 2 was not detected.…”

mentioning

confidence: 99%

“…Mutagenesis and footprinting studies indicate that the two ExsA monomers bind to promoter regions in the same orientation (head-to-tail) at adjacent binding sites which are centered at the Ϫ44 (binding site 1) and Ϫ65 (binding site 2) positions relative to the transcriptional start site (3,12). This binding arrangement is typical of class II activators, which bind in the forward orientation near the Ϫ35 region and typically recruit RNA polymerase holoenzyme through interactions with region 4.2 of the sigma subunit (2,13,14).…”

mentioning

confidence: 99%

“…The second helix in each HTH motif functions as a recognition helix that inserts into adjacent major grooves of the DNA to make base-specific contacts with promoter elements separated by ϳ10 bp (sometimes referred to as A-and B-box sequences). The rec-ognition helices of an individual ExsA monomer, one from each HTH motif, are designated RH1 and RH2 (12). Base-specific contacts mediated by RH1 and RH2 have been defined for binding site 1 in the P exoT promoter.…”

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confidence: 99%

“…Nucleotide substitutions in each of these conserved regions of P exoT result in a significant decrease in ExsA His binding in vitro and ExsA-dependent transcription in vivo. Although the role of the adenine-rich region has not been established, the GnC and TGnnA sequences constitute binding site 1 and are recognized by ExsA through interactions mediated by RH1 and RH2, respectively (12). Because the GnC and TGnnA sequences are conserved in binding site 1 of all known ExsA-dependent promoters, it has been proposed that the mechanistic basis for occupation of binding site 1 is similar at each promoter (12).…”

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confidence: 99%

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The Distal ExsA-Binding Site in Pseudomonas aeruginosa Type III Secretion System Promoters Is the Primary Determinant for Promoter-Specific Properties

Brutinel

King

Marsden

et al. 2012

Journal of Bacteriology

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Section: Resultsmentioning

confidence: 93%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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The Distal ExsA-Binding Site in Pseudomonas aeruginosa Type III Secretion System Promoters Is the Primary Determinant for Promoter-Specific Properties

Brutinel

King

Marsden

et al. 2012

Journal of Bacteriology

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In vitro Assays to Monitor the Activity of Pseudomonas aeruginosa Type III Secreted Proteins

Rolsma

Frank

2014

Methods in Molecular Biology

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Pseudomonas aeruginosa secretes numerous toxins and destructive enzymes that play distinct roles in pathogenesis. The Type III secretion system (T3SS) of Pseudomonas is a system that delivers a subset of toxins directly into the cytoplasm of eukaryotic cells. The secreted effectors include ExoS, ExoT, ExoU, and ExoY. In this chapter, we describe methods to induce T3S expression and measure the enzymatic activities of each effector in in vitro assays. ExoU is a phospholipase and its activity can be measured in a fluorescence-based assay monitoring the cleavage of the fluorogenic substrate, PED6. ExoS and ExoT both possess ADP-ribosyltransferase (ADPRT) and GTPase-activating protein (GAP) activity. ADPRT activity can be assessed by using radiolabeled nicotinamide adenine dinucleotide (NAD+) and measuring the covalent incorporation of ADP-ribose into a target protein. GAP activity is measured by the release of radiolabeled phosphate from [γ-32P]GTP-bound target proteins. In accordance with recent trends towards reducing the use of radioactivity in the laboratory, alternative assays using fluorescent or biotin-labeled reagents are described. ExoY is a nucleotidyl cyclase; cAMP production stimulated by ExoY can be monitored using reverse-phase HPLC or with commercially available immunological assays.

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Determining the Architecture of a Protein–DNA Complex by Combining FeBABE Cleavage Analyses, 3-D Printed Structures, and the ICM Molsoft Program

James

Hsieh

Knipling

et al. 2015

Methods in Molecular Biology

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Determining the structure of a protein-DNA complex can be difficult, particularly if the protein does not bind tightly to the DNA, if there are no homologous proteins from which the DNA binding can be inferred, and/or if only portions of the protein can be crystallized. If the protein comprises just a part of a large multi-subunit complex, other complications can arise such as the complex being too large for NMR studies, or it is not possible to obtain the amounts of protein and nucleic acids needed for crystallographic analyses. Here, we describe a technique we used to map the position of an activator protein relative to the DNA within a large transcription complex. We determined the position of the activator on the DNA from data generated using activator proteins that had been conjugated at specific residues with the chemical cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). These analyses were combined with 3-D models of the available structures of portions of the activator protein and B-form DNA to obtain a 3-D picture of the protein relative to the DNA. Finally, the Molsoft program was used to refine the position, revealing the architecture of the protein-DNA within the transcription complex.

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Orientation of Pseudomonas aeruginosa ExsA Monomers Bound to Promoter DNA and Base-Specific Contacts with the PexoT Promoter

Cited by 18 publications

References 46 publications

The Distal ExsA-Binding Site in Pseudomonas aeruginosa Type III Secretion System Promoters Is the Primary Determinant for Promoter-Specific Properties

The Distal ExsA-Binding Site in Pseudomonas aeruginosa Type III Secretion System Promoters Is the Primary Determinant for Promoter-Specific Properties

In vitro Assays to Monitor the Activity of Pseudomonas aeruginosa Type III Secreted Proteins

Determining the Architecture of a Protein–DNA Complex by Combining FeBABE Cleavage Analyses, 3-D Printed Structures, and the ICM Molsoft Program

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