Figure 1: Multiple reporters allow the simultaneous investigation of independent interactions by bacterial hybrid assay.

(a) Rearrangement of the original B1H reporter vector to express the selectable HIS3 and URA3 markers from separate promoters allows the assay of two independent interactions simultaneously. The addition of fluorescent markers mCherry and GFP provides a secondary, graded measure of activity for each interaction. (b) Survival is dependent on the affinity of the protein–DNA interaction and the selection conditions that impact each reporter independently. Here Zif268 is expressed as an omega fusion. Zif268’s consensus binding site (labelled a) is placed in front of the URA3 reporter and paired with one of a set of binding sites of known affinity in front of HIS3 (numbered from 1 to 6 by the noted ‘X’-fold decrease in affinity offered by each site⁵¹). As a control, a sequence Zif268 will not bind to (labelled b) is placed in front of URA3 and paired with the consensus in front of HIS3. Log-phase cells were titred in 10-fold dilutions from top to bottom on rich media or selective plates that contain 6-azauracil and either a low (2 mM), medium (5 mM) or high (20 mM) level of 3-AT. Survival is dependent on activation of URA3 (1a versus 1b) and related to the affinity of the interaction that drives HIS3 expression. (c) Fluorescent output is related to the affinity of the protein–DNA interaction that drives its expression and unrelated to the selection conditions impacting the competing binding site and reporter. The same cells tested in c, as well as a complete set of the site 1 sequences paired with the negative ‘b’ sequence, were grown in either rich media or selective media that impacts only URA3 expression. While HIS3 expression is not selected for, GFP output is related to the affinity of the interaction that drives the HIS3/GFP reporter and unrelated to the growth conditions that impact site 2 and URA3/mCherry.