Abstract
The evolution of transcriptional regulators through the recruitment of DNA-binding domains by enzymes is a widely held notion. However, few experimental approaches have directly addressed this hypothesis. Here we report the reconstruction of a plausible pathway for the evolution of an enzyme into a transcriptional regulator. The BzdR protein is the prototype of a subfamily of prokaryotic transcriptional regulators that controls the expression of genes involved in the anaerobic degradation of benzoate. We have shown that BzdR consists of an N-terminal DNA-binding domain connected through a linker to a C-terminal effector-binding domain that shows significant identity to the shikimate kinase (SK). The construction of active synthetic BzdR-like regulators by fusing the DNA-binding domain of BzdR to the Escherichia coli SKI protein strongly supports the notion that an ancestral SK domain could have been involved in the evolutionary origin of BzdR. The loss of the enzymatic activity of the ancestral SK domain was essential for it to evolve as a regulatory domain in the current BzdR protein. This work also supports the view that enzymes precede the emergence of the regulatory systems that may control their expression.
Highlights
Regulation of transcription through the action of small molecules that directly bind to a transcription factor is widespread in all life forms
In vivo experiments revealed that the addition of shikimate to the culture medium of E. coli cells expressing the PN::lacZ reporter fusion and the Q2 chimera reduced the repression exerted by Q2 (Figure 3C), a behaviour not observed for the Q1 chimera. These results indicate that when the PN promoter is repressed by the Q2 chimera it can be de-repressed in vivo by shikimate.This observation is consistent with the fact that the catalytically inactive shikimate kinase (SK) domain of the synthetic Q2 regulator can adopt a permanent loaded-like conformation under physiological conditions when shikimate is present, triggering the release of the repressor from the target promoter
We suggest that SK behaves as a ‘‘stem’’ protein domain, able to interact with different substrates and facilitating the acquisition of new functions over time
Summary
Regulation of transcription through the action of small molecules that directly bind to a transcription factor is widespread in all life forms. A large number of transcriptional regulators contain a DNA-binding domain fused to an effector-binding domain. The effector-binding protein domains of transcriptional regulators appear to have evolved by distinct selective forces. The effector-binding protein domains appear to derive from catalytic proteins, which may or may not retain the active site residues in their binding pockets during evolution and could possibly behave as bifunctional proteins [2]. There are a few examples of transcriptional regulators that might have evolved from enzymes that have lost their catalytic activity. The HutC regulator of Pseudomonas putida, involved in histidine utilization [5], and the FarR regulator of Escherichia coli, which controls the expression of Krebs cycle genes and responds to fatty acids [6], contain effector-binding domains similar to chorimaste lyases [2]. The evolutionary pathways giving rise to these regulators have not yet been reproduced in the laboratory
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