Evolutionary conservation and arrangement of domains in the RNAP active site. (A) Most structures are shown in space-filling mode to emphasize spatial connections. The Bridge Helix is shown in green, with the regions subjected to high-throughput mutagenesis in this study highlighted in yellow. The template DNA is pale blue, the RNA is red, the NTP in the insertion site is shown as a pink stick model and catalytic metal ions as magenta spheres. Three additional domains that surround the Bridge Helix N-terminus, β-D II domain (turquoise) and Link (light purple) and the Trigger Loop (dark blue cartoon) are shown. (PDB #2E2H). (B) Alignment of Bridge Helix sequences from bacteria (Escherichia coli K12 (Genbank BAE77332); Thermus aquaticus (Genbank RPOC_THEAQ); Thermus thermophilus (Genbank RPOC_THET8) and eukaryotes (Saccharomyces cerevisiae (Genbank CAA26904) and Homo sapiens (Genbank EAW90183)) against the archaeon M. jannaschii (Genbank A64430). Residues identical to the archaeal sequence are shown in red. The region mutagenized is highlighted with yellow background. The residues contacted by α-amanitin (eukaryotic RNAPII) and streptolydigin (bacterial RNAPs) are boxed. The numbers flanking the sequences represent the location of the sequences within the open reading frame of the complete subunit.