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Figure 1 | Journal of Biology

Figure 1

From: Bridge helix and trigger loop perturbations generate superactive RNA polymerases

Figure 1

Structure, evolutionary conservation and conformational isomers of the bridge helix. (a) Structure of the active site of Saccharomyces cerevisiae RNAPII [7] (based on PDB code 2E2H). All structures, except the trigger loop (dark blue) and the rNTP in the insertion site (salmon pink) are shown in the space-filling representation. The bridge helix is green and the region that has been mutagenized for this study is highlighted in yellow. The DNA template strand is in light blue and the nascent transcript red. The Mg2+ ion (metal 'A', magenta) is part of the catalytic site. (b) Sequence alignment of representative bacterial [Escherichia coli K12 (UniProt/Swiss-Prot accession number P0A8T7), Thermus aquaticus (Q9KWU6), Thermus thermophilus HB27 (Q72HM6)], archaeal [Methanocaldococcus jannaschii (A64430) and Sulfolobus solfataricus (NP_341776)] and eukaryotic [S. cerevisiae (CAA65619), Homo sapiens (NP_000928); RNAPII only] bridge helix sequences. Except for H. sapiens and M. jannaschii, all other sequences have been selected solely on the basis of the availability of X-ray structures. The numbers on the left and right side of the sequences refer to the amino- and carboxy-terminal amino acid positions of the sequence shown relative to the full-length open reading frame. The numbering of the residues in the M. jannaschii sequence is based on the intein-free final product. Amino acid residues identical to the corresponding M. jannaschii position are shown in red, the 'hinge' region [11, 12] in gray and binding sites of α-amanitin [13, 19] and streptolydigin [12, 20] are boxed in blue. Note that the location of the hinge region in archaeal and eukaryotic RNAPs is inferred from the position of the kink in bacterial bridge helices. The residues in the M. jannaschii sequence that were mutagenized in this study are highlighted with a yellow box. (c) Top view (from the amino terminus of the bridge helix) of structure shown in (a). Note the position of the bridge helix relative to the DNA-RNA hybrid. (d) Bridge helix conformations as seen in three different X-ray structures. The bridge helix is shown in green in ribbon representation. The species and PDB codes are shown below. Two orthologous residues in each structure, corresponding to D1090 and R1096 in the T. thermophilus β' subunit, are shown in stick representation in red and blue, respectively (see text for further discussion of the possible significance of these residues in stabilizing the kinked conformation).

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