Extended Data Fig. 6: A model of the gp66 catalytic site in the active conformation. | Nature
a, DNA-dependent RNA synthesis activity of wild-type gp66 and its two catalytic loop mutants as measured on denatured phi14:2 DNA. The uncropped autoradiogram is shown in Supplementary Fig. 5. The assay was performed three times for each of two biological replicates. b, The crystal structure of the catalytic site of the T. thermophilus RNAP (the elongation state conformation, PDB ID: 2O5J15). Residues of the β subunit are labelled with a superscript index. c, Possible configuration of the catalytic site of the wild-type gp66 (tan coloured) and its I1364G and I1364W mutants (coloured cyan and magenta, respectively) in the active conformation. The models were obtained by rebuilding and regularizing the geometry of the polypeptide chain to make the side chains of the three catalytic aspartates (D1361, D1363 and D1365) point in the same direction. For each of the aspartates, a rotamer that brings the side chains closer in space was chosen. The magnesium ion was placed to match its dictionary distance value. In b, c, the bottom panels show the Ramachandran angles of the amino acids comprising the catalytic loop (the Ramachandran plots of gp66 and its two mutants are overlaid on top of each other); residues that interact with the catalytic loop are semi-transparent and represent the crystal structure conformation.