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. 2010 Jun 18;29(15):2527–2537. doi: 10.1038/emboj.2010.135

Figure 4.

Figure 4

Lpm blocks formation of the lacUV5-promoter-open complex. (A) Kinetic steps in transcription initiation at the lacUV5 promoter (Buc and McClure, 1985). P—promoter, R—RNAP, RPc—closed complex, RPi—intermediate complex, RPo—open complex. (B) EMSA of the complexes of RNAP (WT—wild-type, R337A—mutant RNAPR337A) and fluorescein-labelled lacUV5-promoter fragment formed either in the presence of Lpm or heparin or both. Complexes were resolved in native 5% PAGE. The position of the open complex (RPo), closed complex (RPc) and non-bound DNA (Free DNA) are shown. (C) KMnO4 probing of the lacUV5-promoter complexes formed by the wild-type RNAP (WT) and mutant RNAPR337A (R337A) in the presence of 50 μM of Lpm (lanes 2, 3, 5 and 6). Star indicates Lpm added after the RPo formation (lanes 3 and 6). DNA was labelled by fluorescein at the 5′ end of template strand. The positions of thymines reactive to KMnO4 in the open complex are indicated. M—A+G sequencing marker. Profiles on the right show the scan of the lanes for RPo (red) and RPo+Lpm (green). (D, E) DNAse I footprinting of the lacUV5-promoter complexes in the presence of 100 μM of Lpm. DNA labelled either at non-template (D) or template (E) strands. Profiles on the right show the scan of the lanes for free DNA (blue), RPo (red) and RPo+Lpm (green). (F) The bar graph shows quantification of the DNAse I footprinting result for template DNA strand. The ratio of peak area values for indicated bands within the promoter region protected by RNAP in RPo in the presence or without Lpm were normalized to the peak area values for corresponding bands of free DNA. [T-45] and [T-35] designate the sum of signals in the positions −45 to −39 and the positions −34 to −30 correspondingly. The peak areas values of the bands within the each lane were normalized to the area values of the bands not protected by RNAP within the same lane (positions −48, −50). An average and standard errors of two experiments are shown. The quantified bands are indicated by the red stars in panel E. The RNAP holoenzyme domains interacting with the DNA bases at the indicated positions of promoter in RPo are shown schematically beneath the bar graph.