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. 2014 Jul 31;42(15):9677–9690. doi: 10.1093/nar/gku690

Figure 3.

Figure 3.

(A) Autoradiograms showing the in vitro transcription termination assays on two types of linear DNA templates having trac and tR1 terminators under different conditions as indicated. Run-off (RO) products and terminated products (dashed lines) are indicated. ‘*’ is an arrested product characteristic of this template. Molecular weight markers (M) are indicated adjacent to the autoradiograms. (B) Schematic showing constructs where different terminators, tR1, trpt′ and trac, were fused upstream of the lacZ reporter. An untranslated region, having putative Rho-loading site(s) (tybck; see Supplementary Figure S8A), is located upstream of ybcK. LacZ reporter cassettes were inserted in the chromosome of MC4100 strain by λRS45 mediated transduction. Arrows indicate primer pairs used in RT-PCR reactions. (C) NusG-CTD point mutants defective for Rho-binding are shown on the structure (12). (D) Bar diagrams showing the β-galactosidase activities obtained from the indicated terminator-lacZ fusions in the presence of WT and different NusG mutants expressed from pHyd311 plasmid. Y-axis has been shown as a broken scale to accommodate the full range of values. Errors were obtained by measuring the activities of six independent colonies. The activities in the presence of WT NusG were set at 1, and the fold change values in the presence of NusG mutants were expressed with respect to the WT value. The raw data of β-galactosidase activities are described in Supplementary Table S2. (E) EtBr stained 1.5% agarose gel showing the RT-PCR products obtained from the lacZ gene fused to tR1 terminator (left panel) and from the ybcK (right panel) using appropriate primer pairs indicated in (B). RNA was isolated from the strains expressing indicated WT or mutant NusG supplied from pHYD311. In a similar way as above, the amount of RT-PCR products (intensity of the bands) was also expressed as fold change with respect to WT NusG. Intensities of the bands were measured using Image J, a version of NIH Image for Personal Computers (PCs). (F) Growth characteristic of MC4100rac−Δrho nusGG146D or nusGL158Q or WT nusG upon transformations with pCL1920 plasmids expressing either WT or N340S or Y80C Rho mutants. Two transductants of each strain were re-streaked on LB plates.