Figure 5.
Functional dissection of the BH-HC region. (A) Position of mutagenized residues. The residues that differ from the archaeal wildtype sequence in the various mutagenesis constructs are shown in red. Wildtype sequences of the M. jannaschii and S. cerevisiae Bridge Helix sequences are shown for comparison. The position of BH-HC is marked with an arrow. (B) The center of the panel provides an overview depicting the orthologous residues in space-filling mode within the mutagenized part of the Bridge Helix modelled on the yeast RNAPII structure (PDB #2E2H); yeast residues E823 and T824 were replaced in silico with Q and P, respectively, to reveal the approximate location of these amino acids relative to the DNA-RNA hybrid and catalytic site). Adjacent parts of the Bridge Helix domain are shown as a transparent ribbon. All other colors are coded as in Figure 1A (template DNA is pale blue, the nascent transcript is red, the NTP in the insertion site is pink and catalytic metal ions are shown as magenta spheres). All mutants shown here contain the mjA' S824-P substitution (yellow). The results of promoter-independent activity assays are plotted relative to wildtype activity (indicated with a red dashed line). Two of the bar charts show the effect of introducing systematic substitutions in positions located immediately N- or C-terminal to S824-P (mjA' Q823 [lime green; upper right]; mjA' G825 [orange; lower left]). The other two bar charts show the functional consequences of introducing additional systematic substitutions in positions located immediately N-terminal to the double proline substitution Q823-P/S824-P (mjA' A822 [olive green; upper left]), or immediately C-terminal to the double proline substitution S824-P/G825 (mjA' Y826 [brown; lower left]). The colors of the histogram bars match the colors of the substituted residues in the structural model. All assays were performed in at least quadruplicate, with error bars showing standard deviation from the average value.