Abstract
The biophysical nature of the interaction between a transcription factor and its target sequences in vitro is sufficiently well understood to allow for the effects of DNA sequence alterations on affinity to be predicted. But even in relatively simple in vivo systems, the complexities of promoter organization and activity have made it difficult to predict how altering specific interactions between a transcription factor and DNA will affect promoter output. To better understand this, we measured the relative fitness of nearly all Escherichia coli binding sites in different promoter and environmental contexts by competing four randomized promoter libraries controlling the expression of the tetracycline resistance gene (tet) against each other in increasing concentrations of drug. We sequenced populations after competition to determine the relative enrichment of each −35 sequence. We observed a consistent relationship between the frequency of recovery of each −35 binding site and its predicted affinity for that varied depending on the sequence context of the promoter and drug concentration. Overall the relative fitness of each promoter could be predicted by a simple thermodynamic model of transcriptional regulation, in which the rate of transcriptional initiation (and hence fitness) is dependent upon the overall stability of the initiation complex, which in turn is dependent upon the energetic contributions of all sites within the complex. As implied by this model, a decrease in the free energy of association at one site could be compensated for by an increase in the binding energy at another to produce a similar output. Furthermore, these data show that a large and continuous range of transcriptional outputs can be accessed by merely changing the , suggesting that evolved or engineered mutations at this site could allow for subtle and precise control over gene expression.
Highlights
While we have a reasonable understanding of the biophysical forces that determine the affinity of a transcription factor to its target sequences [1,2,3,4], we still have a poor understanding of how the affinity of a factor for a site affects the output of the promoter in which it sits
A high affinity site tightly bound in isolation will have no function in that it will not affect the rate of transcription of a gene, whereas a low affinity site weakly bound in the context of the initiation complex will
In the simplest thermodynamic model of transcriptional regulation in prokaryotes, the rate of transcriptional output varies as a direct function of the stability of the initiation complex [8,9,10,11]
Summary
While we have a reasonable understanding of the biophysical forces that determine the affinity of a transcription factor to its target sequences [1,2,3,4], we still have a poor understanding of how the affinity of a factor for a site affects the output of the promoter in which it sits. The general assumption has been that the greater the affinity that the factor has for a site, the greater the occupancy at that site and the greater the probability that it will affect transcription [10] This has only recently been tested for large libraries of sequences, and much of the variance in expression can be explained by differences in binding site affinity [12]. Given this relationship, the distribution of binding energies for a factor defines the range of regulatory phenotypes that can be Author Summary
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