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GENETICS AND MOLECULAR BIOLOGY

The Escherichia coli Ada Protein Can Interact with Two Distinct Determinants in the ς70 Subunit of RNA Polymerase According to Promoter Architecture: Identification of the Target of Ada Activation at the alkAPromoter

Paolo Landini, Stephen J. W. Busby
Paolo Landini
School of Biochemistry, The University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
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Stephen J. W. Busby
School of Biochemistry, The University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
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DOI: 10.1128/JB.181.5.1524-1529.1999
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    Fig. 1.

    In vivo transcription from the alkA promoter. The effects of the substitutions in ς70 region 4 onalkA expression are shown as percentages of alkAexpression with wild-type ς70. Values (± standard deviations) are averages of four independent experiments. The average value for the wild type was 342 Miller units.

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    Fig. 2.

    In vitro transcription with reconstituted RNA polymerases (50 nM). Lanes 1 to 3, wild-type Eς70; lanes 4 to 6, Eς70KA593; lanes 7 to 9, Eς70KA597; lanes 10 to 12, Eς70RA603. Lanes 1, 4, 7, and 10, no Ada protein; lanes 2, 5, 8, and 11, 0.2 μM unmethylated Ada; lanes 3, 6, 9, and 12, 0.2 μM methylated Ada. The positions of the mainlacUV5 and alkA transcripts are indicated.

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    Fig. 3.

    Ratio of alkA/lacUV5 transcripts. Values are percentages of transcription by wild-type Eς70 in the presence of meAda and are averages of three independent experiments. Dark grey bars, transcription levels in the absence of Ada; light grey bars, transcription levels in the presence of unmethylated Ada; black bars, transcription levels in the presence of methylated Ada. Standard deviations were less than 15%.

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    Fig. 4.

    Gel retardation assays performed with reconstituted RNA polymerase. Odd-numbered lanes, no meAda; even-numbered lanes, 0.2 μM meAda. Lanes 1 and 2, no RNA polymerase; lanes 3 and 4, wild-type Eς70; lanes 5 and 6, Eς70KA593; lanes 7 and 8, Eς70KA597; lanes 9 and 10, Eς70RA603. F, unbound alkApromoter DNA; C, RNA polymerase-alkA complex.

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    Fig. 5.

    In vitro transcription with Eς70 (lanes 1 to 3) and EςS (lanes 4 to 6) forms of RNA polymerase. Lanes 1 and 4, no Ada protein; lanes 2 and 5, 0.2 μM Ada; lanes 3 and 6, 0.2 μM meAda. The positions of the mainlacUV5 and alkA transcripts are indicated.

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    Fig. 6.

    Gel retardation assays performed with Eς70and EςS forms of RNA polymerase. Odd-numbered lanes, nomeAda; even-numbered lanes, 0.2 μM meAda. Lanes 1 and 2, no RNA polymerase; lanes 3 and 4, Eς70; lanes 5 and 6, EςS. F, unbound alkA promoter DNA; C70 and CS, Eς70-alkA and EςS-alkA complexes, respectively.

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The Escherichia coli Ada Protein Can Interact with Two Distinct Determinants in the ς70 Subunit of RNA Polymerase According to Promoter Architecture: Identification of the Target of Ada Activation at the alkAPromoter
Paolo Landini, Stephen J. W. Busby
Journal of Bacteriology Mar 1999, 181 (5) 1524-1529; DOI: 10.1128/JB.181.5.1524-1529.1999

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The Escherichia coli Ada Protein Can Interact with Two Distinct Determinants in the ς70 Subunit of RNA Polymerase According to Promoter Architecture: Identification of the Target of Ada Activation at the alkAPromoter
Paolo Landini, Stephen J. W. Busby
Journal of Bacteriology Mar 1999, 181 (5) 1524-1529; DOI: 10.1128/JB.181.5.1524-1529.1999
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KEYWORDS

Bacterial Proteins
DNA Glycosylases
DNA-Directed RNA Polymerases
Escherichia coli
Escherichia coli Proteins
N-Glycosyl Hydrolases
sigma factor

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