Figure 5.

Activity assays of trigger loop mutants. (a) Graphical overview ('heat map') of the mutant activities from high-throughput non-specific transcription assays. The vertical axis shows the identity of the residues located along the M. jannaschii trigger loop. On the horizontal axis the amino acid substitutions for each of these positions is marked. The transcriptional activities of the mutants are color-coded according to the scale shown in the right relative to the wild-type activity (defined as 100%). The activity values for each substitution are based on a minimum of four independent assemblies/transcription assays. (b) Prediction of local stabilities of substitutions in the mjA'' I98 position. The 'FoldIndex' was calculated using a web-based program, FoldIndex [48] (window size = 3; step = 1) with mjA'' amino acid sequences containing substitutions with all 20 different amino acids in the I98 position. Areas within the graph with positive FoldIndex values (stable folding) and negative values (unstable folding) are indicated by a light green or magenta background, respectively. I98 seems to occupy a critical area between regions of low stability (R96) and high stability (E99 and I100). The identity of residue 98 (highlighted between the dashed lines) critically determines the shape of this transition; a small number of hydrophobic residues increases local stability (I>V>L>F>C>M>A; green symbols), whereas most other substitutions cause local destabilization (for example P, D or E; magenta). Certain substitutions may cause additional structural changes that cannot be accounted for by this prediction. The symbols for the various amino acids are shown on the right.