Cauã Antunes Westmann, Leander Goldbach, Andreas Wagner
Transcription factor binding sites (TFBSs) are important sources of evolutionary innovations. Understanding how evolution navigates the sequence space of such sites can be achieved by mapping TFBS adaptive landscapes. In such a landscape, an individual location corresponds to a TFBS bound by a transcription factor. The elevation at that location corresponds to the strength of transcriptional regulation conveyed by the sequence. Here, we develop an in vivo massively parallel reporter assay to map the landscape of bacterial TFBSs. We apply this assay to the TetR repressor, for which few TFBSs are known. We quantify the strength of transcriptional repression for 17,765 TFBSs and show that the resulting landscape is highly rugged, with 2092 peaks. Only a few peaks convey stronger repression than the wild type. Non-additive (epistatic) interactions between mutations are frequent. Despite these hallmarks of ruggedness, most high peaks are evolutionarily accessible. They have large basins of attraction and are reached by around 20% of populations evolving on the landscape. Which high peak is reached during evolution is unpredictable and contingent on the mutational path taken. This in-depth analysis of a prokaryotic gene regulator reveals a landscape that is navigable but much more rugged than the landscapes of eukaryotic regulators.
Cauã Antunes Westmann, Leander Goldbach, Andreas Wagner. The highly rugged yet navigable regulatory landscape of the bacterial transcription factor TetR. 2024-12-30. https://doi.org/10.1038/s41467-024-54723-y
An eight-position combinatorial tetO2 transcription-factor-binding-site library (4^8 = 65,536 theoretical variants) was cloned into a low-copy plasmid reporter in Escherichia coli. TetR-mediated repression was measured by GFP fluorescence after FACS sorting into 13 bins and amplicon sequencing; the processed table contains the 17,765 variants in the QC-filtered giant component used for landscape analyses.
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