Mutations in the tyrR gene of Escherichia coli which affect TyrR-mediated activation but not TyrR-mediated repression

Abstract

Site-directed mutagenesis has been used to further characterize amino acid residues necessary for the activation of gene expression by the TyrR protein. Amino acid substitutions have been made at positions 2, 4, 5, 6, 7, 8, 9, 10, and 16. TyrR mutants with amino acid substitutions V-5-->P (VP5), VF5, CS7, CR7, DR9, RI10, RS10, and ER16 show no or very little activation of expression of either mtr or tyrP. In each case, however, the ability to repress aroF is unaltered. Amino acid substitutions at positions 4, 6, and 8 have no effect on activation. Small internal deletions of residues 10 to 19, 20 to 29, or 30 to 39 also destroy phenylalanine- or tyrosine-mediated activation of mtr and tyrP. In these mutants repression of aroF is also unaltered. In activation-defective tyrR mutants, expression of mtr is repressed in the presence of tyrosine. This tyrosine-mediated repression is trpR dependent and implies an interaction between TrpR and TyrR proteins in the presence of tyrosine.

Further genetic analysis of the activation function of the TyrR regulatory protein of Escherichia coli

“…Mutants PT90, DC97, and DF103 showed enhanced activation from both the mtr and the tyrPϩ4 promoters (Table 2). In the case of mutant LF95, the activation efficiency was slightly decreased at the mtr promoter and unchanged at the tyrPϩ4 promoter (Table 2) viously that substitutions of E-4 and E-8 with neutral amino acids such as in EL4 and EF8 showed practically no change in activation (25). On the other hand, substitution of a positively charged arginine for either aspartate 9 (DR9) or glutamate 16 (ER16) resulted in a significant reduction in the activation function of TyrR.…”

“…Previously we have made three internal deletions within TyrR which removed residues 10 to 19, 20 to 29, or 30 to 39, and we showed that proteins with each of these deletions are defective in phenylalanine-and tyrosinemediated activation of mtr and tyrPϩ4 (25). To further characterize the activation domain of TyrR, we used site-directed mutagenesis to construct four new internal deletions of residues 40 to 49, 50 to 59, 60 to 69, and 90 to 99 ( Fig.…”

“…This indicated that they included one RQ10, one CR7, two with a change of glycine to arginine at position 37 (GR37), one with a change of glycine to glutamate at position 37 (GE37), one with a change of methionine to isoleucine at position 55 (MI55), and one with two mutations, GR37 and a change of aspartate to valine at position 9 (DV9). The mutations RQ10 (this paper) and CR7 (25) had already been identified as causing loss of activation, and position 9 was also known to be important (25), so these three mutants (RQ10, CR7, and DV9-GR37) were not studied further. The three new mutants, GR37, GE37, and MI55 (Fig.…”

“…Functional mapping by deletion mutagenesis has located the activation function to the N-terminal domain (6,26). By making specific amino acid substitutions, we (25) and others (5) have identified a number of amino acid residues at positions 2, 3, 5, 7, 9, 10, and 16 as playing an important role in activation. As previous functional mapping of TyrR was carried out by site-directed mutagenesis and was limited to the N-terminal region, two questions remained to be answered: (i) is the Nterminal domain the only region which is directly involved in activation, and (ii) are there any other amino acid residues in the N-terminal domain which are essential for activation?…”

The various strategies within the TyrR regulon of Escherichia coli to modulate gene expression

“…Work from two laboratories has established that the amino-terminal domain is essential for the activation of expression of the genes mtr and tyrP (Cui & Somerville 1993a,b;Yang et al 1993aYang et al ,b, 1996a. Much of this domain can be deleted without affecting the activity of TyrR as a repressor, and a number of mutants with single amino acid substitutions, which can no longer activate gene expression but which retain full activity as repressors, have been described.…”