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TyrR

Part Description

The TyrR protein in Escherichia coli has the ability to both activate and repress the transcription operons necessary for the production and intake of aromatic amino acids (tyrosine, phenylalanine, and tryptophan). As an illustration, the TyrR dimer activates the promoter of the tyrP gene, which codes for a transporter specific to tyrosine, when tyrosine is present

Usage

We took advantage of The TyrR protein properties and it’s ability to control the biosynthesis and intake of aromatic amino acids (tyrosine, phenylalanine, and tryptophan). So we employ it to control the activity of the paroF promoter and TyrP promoter as TyrR dimer activates the TyrP promoter and paroF promoter in the presence of phenylalanine , when tyrosine is more abundant the induction effect on paroF is lost

Literature Characterization

ATPase activity of tyrR:
In this study, TyrR has poor ATPase activity that ranges between 12 and 400 mmol of ATP mol−1 monomer min−1. TyrR-(188–467) posess a specific ATPase activity of 105 mmol of ATP mol−1 monomer min−1, about five times the value for TyrR, which was found in this study to be 20 mmol of ATP mol−1 monomer min−1. As shown in figure 1, the flow dialysis demonstrates that TyrR binds to ATP with a half saturation value of 3.1 m. TyrR-(188-467) binds to ATP with a half saturation value of 7.6 m. TyrR-(188-467) had a 1.4 m dissociation constant when it coupled to rhodamine-ATP. These findings demonstrate that TyrR-(188-467) had a 2- to 5-fold reduced affinity for binding ATP and rhodamine-ATP than TyrR. Dixon, M. P., Pau, R. N., Howlett, G. J., Dunstan, D. E., Sawyer, W. H., & Davidson, B. E. (2002). The central domain of Escherichia coli TyrR is responsible for hexamerization associated with tyrosine-mediated repression of gene expression. Journal of Biological Chemistry, 277(26), 23186-23192.‏

Figure 1. Flow dialysis analysis of ATP binding to TyrR and TyrR-(188–467). The fractional saturation with ATP of 40 μM TyrR (closed circles) and TyrR-(188–467) (open circles) at various concentrations of ATP

Characterization of Mutational Landscape

After creating a multiple sequence alignment of the protein sequence and predicting mutational landscapes, the effect of these mutations on the evolutionary fitness of the protein is tested. The prediction of the mutational landscape by saturation mutagenesis of the TyrR protein. The (C374R) mutation, as depicted in the chart, had the greatest score when compared to other mutations. On the other hand, it's clear that the (K399Q) had the least evolutionary fitness for TyrR protein. As displayed in Figure(2)

Figure 2. (shows the mutational landscape of TyrR protein)

References

1. Dixon, M. P., Pau, R. N., Howlett, G. J., Dunstan, D. E., Sawyer, W. H., & Davidson, B. E. (2002). The central domain of Escherichia coli TyrR is responsible for hexamerization associated with tyrosine-mediated repression of gene expression. Journal of Biological Chemistry, 277(26), 23186-23192.‏ Sequence and Features