Figure 2.

Activity assays of bridge helix mutants. (a) Graphical overview ('heat map') of the mutant activities from high-throughput non-specific transcription assays. The vertical axis shows the identity of the residues located along the M. jannaschii bridge helix, spanning the interval from L814 to R830 (inclusive). On the horizontal axis the amino acid substitutions for each of these positions is indicated. The specific transcriptional activities of the mutants are color-coded according to the scale shown lower right, ranging from inactive (dark blue, 0%) to superactive (dark red, 200%) relative to the wild-type activity (defined as 100%). The activity values for each substitution are based on a minimum of four independent assemblies and transcription assays (see Additional data files 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 for further details). Data for the mjA' G825 substitutions have been published previously [25] but are included here for completeness. (b) Polar plot ('helical wheel') of mutant activities reflecting the spatial arrangement of the residues relative to each other in the α-helical bridge helix. The activities of substitutions in individual residues (as labeled on the periphery) are plotted along the radius. Activities below the wild-type level (100%) are in black, whereas activities above that level are coded by their color and radial position. The figures along the 90°, 180°, 270° and 0/360° axes refer to percentage of wild-type activity. (c) Abortive transcription assays showing the incorporation of [α-³²P]rUTP into abortive dinucleotide extension products on activated DNA during a 20-minute incubation period. (d) Multiple-round elongation transcription assays on a DNA-RNA scaffold. The position of the extension product is marked FL.