Figure 2.

High-throughput mutagenesis of the Bridge Helix. (A) The specific activities in recruitment-independent transcription assays of systematic substitutions of archaeal Bridge Helix residues H806 to R830 are shown as a heat map relative to the activity of the wildtype enzyme (see adjacent scale for comparison). Substitutions in M808 cause an exceptional increase in catalytic activity with chemically diverse side chains. Previously published data (L814 to R830; [20]) are included to provide context. All assays were performed in at least quadruplicate with standard deviations within 12% of the average value. (B) Plot of proline substitutions across the Bridge Helix. The wildtype activity level (100%) is marked with a dashed red line. The substituted residues in the M. jannaschii Bridge Helix are shown along the horizontal axis. Most proline substitutions cause a severe reduction in the specific activity, except at positions M808 and S824, where proline substitutions cause superactivity. All assays were performed in at least quadruplicate, with error bars showing standard deviation from the average value. (C) Naturally occurring proline-substitutions (highlighted in boxes). The Bridge Helix sequences of three bacterial species, Orientia tsutsugamushi (Genbank YP_001248195 (Boryong)/YP_001938485 (Ikeda)), isolates of Arcobacter butzleri (Genbank AAZ80810) and Bacillus subtilis (Genbank BAA10999), as well as representative examples of plant RNAP IV and V Bridge Helix sequences from Arabidopsis thaliana (Genbank AAY89363 and NP_181532, respectively) and Oryza sativa (Genbank EEE70198 and EEE56320, respectively) are aligned against the M. jannaschii sequence (Genbank A64430). The bacterial sequences each contain a single proline residue corresponding to mjA' M808, whereas proline substitutions in RNAP IV and V align with mjA' S824. Residues identical to the archaeal sequence are shown in red.

Weinzierl BMC Biology 2010 8:134   doi:10.1186/1741-7007-8-134
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