Mutational analysis of repression and activation of the tyrP gene in Escherichia coli.

In a previous report it had been suggested that the tyrP gene of Escherichia coli may be expressed from two separate promoters. We have endeavored to confirm this suggestion by primer extension studies and the separate subcloning of each of these promoters. In these studies, we found a single promoter whose expression was repressed by TyrR protein in the presence of tyrosine and activated by TyrR protein in the presence of phenylalanine. Two adjacent TYR R boxes, with the downstream one overlapping the tyrP promoter, are the likely targets for the action of TyrR protein. Mutational analysis showed that both TYR R boxes were required for tyrosine-mediated repression but that only the upstream box was required for phenylalanine-mediated activation. In vitro DNase protection studies established that whereas in the absence of tyrosine TyrR protein protected the region of DNA represented by the upstream box, at low TyrR protein concentrations both tyrosine and ATP were required to protect the region of DNA involving the downstream box and overlapping the RNA polymerase binding site.     

Importance of the position of TYR R boxes for repression and activation of the tyrP and aroF genes in Escherichia coli.

Tyrosine-mediated repression of aroF and tyrP was studied by inserting DNA sequences between the two adjacent TYR R boxes which, in each case, overlap the respective RNA polymerase binding sites of these genes. In both cases, repression was greatest when homologous regions of these two TYR R boxes were on the same face of the DNA helix and the boxes were directly adjacent. An insertion of 3 bases was sufficient to abolish repression, which was reestablished as the boxes became separated by one full turn of the helix. These observations, coupled with the results of in vitro DNase I protection studies, supported the hypothesis that the binding of TyrR protein to the downstream boxes required cooperative interaction with TyrR protein already bound to the upstream boxes. In the case of tyrP, moving the upstream box also affected activation. Maximal activation was observed when the box was moved 3 or 12 to 14 residues upstream. Practically no activation was seen at intermediate positions, such as +7 and -4. It is hypothesized that these results indicate positions allowing maximal interaction between TyrR protein bound to the upstream box and RNA polymerase bound to the RNA polymerase binding site.     

Regulation of aroL expression by TyrR protein and Trp repressor in Escherichia coli K-12.

The promoter-operator region of the aroL gene of Escherichia coli K-12 contains three TYR R boxes and one TrpR binding site. Mutational analysis showed that TYR R boxes 1 and 3 are essential for TyrR-mediated regulation of aroL expression, while a fully functional TYR R box 2 does not appear to be essential for regulation. Regulation mediated by the TrpR protein required the TYR R boxes and TrpR site to be functional and was observed in vivo only with a tyrR+ strain. Under conditions favoring the formation of TyrR hexamers, DNase I protection experiments revealed the presence of phased hypersensitive sites, indicative of DNA backbone strain. This suggests that TyrR-mediated repression involves DNA looping. Purified TrpR protein protected the putative TrpR binding site in the presence of tryptophan, and this protection was slightly enhanced in the presence of TyrR protein. This result along with the in vivo findings implies that TyrR and TrpR are able to interact in some way. Inserting 4 bp between TYR R box 1 and the TrpR binding site results in increased tyrosine repression and the abolition of the tryptophan effect. Identification of a potential integration host factor binding site and repression studies of a himA mutant support the notion that integration host factor binding normally exerts a negative effect on tyrosine-mediated repression.     

Repression of the aroP gene of Escherichia coli involves activation of a divergent promoter.

The repression of aroP expression which is mediated by the TyrR protein with phenylalanine, tyrosine, or tryptophan has been shown to be primarily a direct result of TyrR-mediated activation of a divergent promoter, P3, which directs the RNA polymerase away from promoter P1. Evidence which has been presented to support this conclusion is as follows. Repression of P1 does not occur either in vitro or in vivo if wild-type TyrR protein is substituted by the activation-negative mutant RQ10 (with an R-to-Q change at position 10). Repression of P1 is greatly diminished if the P3 promoter is inactivated or if a 5-bp insertion is made between the P3 promoter and the binding sites for TyrR. Repression is also abolished if the promoter strength of P1 is increased or a putative UP element associated with P3 is altered. Repression of the second promoter, P2, still occurs if the wild-type TyrR protein is substituted with RQ10 or EQ274. The tryptophan-mediated repression of aroP does not involve the TrpR protein.     

Specific contacts between residues in the DNA-binding domain of the TyrR protein and bases in the operator of the tyrP gene of Escherichia coli

In the presence of tyrosine, the TyrR protein of Escherichia coli represses the expression of the tyrP gene by binding to the double TyrR boxes which overlap the promoter. Previously, we have carried out methylation, uracil, and ethylation interference experiments and have identified both guanine and thymine bases and phosphates within the TyrR box sequences that are contacted by the TyrR protein (J. S. Hwang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:1051-1058, 1997). In this study, we have used missing contact probing to test the involvement of all of the bases within the tyrP operator in the binding of TyrR. Our results indicate that nearly all the bases within the palindromic arms of the strong and weak boxes are important for the binding of the TyrR protein. Two alanine-substituted mutant TyrR proteins, HA494 and TA495, were purified, and their binding affinities for the tyrP operator were measured by a gel shift assay. HA494 was shown to be completely defective in binding to the tyrP operator in vitro, while, in comparison with wild-Type TyrR, TA495 had only a small reduction in DNA binding. Missing contact probing was performed by using the purified TA495 protein, and the results suggest that T495 makes specific contacts with adenine and thymine bases at the +/-5 positions in the TyrR boxes.     

Effects of molecular crowding on the interaction between DNA and the Escherichia coli regulatory protein TyrR

Fluorescence quenching has been used to measure quantitatively the effects of sucrose and triethylene glycol on the interaction between the Escherichia coli regulatory protein TyrR and a 30-basepair oligonucleotide containing the strong TyrR box of the TyrR operon. It was observed that the apparent binding constant increased in the presence of co-solutes, the dependence of the logarithm of the apparent binding constant on molar concentration being indistinguishable and essentially linear for both co-solutes. This activation of the TyrR-oligonucleotide interaction is attributed to thermodynamic nonideality arising from molecular crowding, an interpretation which is supported by the reasonable agreement observed between the experimental extent of reaction enhancement and that predicted on the statistical-mechanical basis of excluded volume.     

Critical base pairs and amino acid residues for protein-DNA interaction between the TyrR protein and tyrP operator of Escherichia coli.

In Escherichia coli K-12, the repression of tyrP requires the binding of the TyrR protein to the operator in the presence of coeffectors, tyrosine and ATP. This operator contains two 22-bp palindromic sequences which are termed TyrR boxes. Methylation, uracil, and ethylation interference experiments were used to identify the important sites in the TyrR boxes that make contacts with the TyrR protein. Methylation interference studies demonstrated that guanines at positions +8, -5, and -8 of the strong TyrR box and positions +8, -4, and -8 of the weak box are close to the TyrR protein. Uracil interference revealed that strong van der Waals contacts are made by the thymines at position -7 and +5 of the top strands of both strong and weak boxes and that weaker contacts are made by the thymines at positions +7 (strong box) and -5 and +7 (weak box) of the bottom strand. In addition, ethylation interference suggested that the phosphate backbone contacts are located at the end and central regions of the palindrome. These findings are supported by our results derived from studies of symmetrical mutations of the tyrP strong box. Overall, the results confirm the critical importance of the invariant (G x C)(C x G)8 base pairs for TyrR recognition and also indicate that interactions with (T x A)(A x T)7 are of major importance. In contrast, mutations in other positions result in weaker effects on the binding affinity of TyrR protein, indicating that these positions play a lesser role in TyrR protein recognition. Alanine scanning of both helices of the putative helix-turn-helix DNA-binding motif of TyrR protein has identified those amino acids whose side chains play an essential role in protein structure and DNA binding.     

Repression of the aroF promoter by the TyrR repressor in Escherichia coli K-12: role of the ''upstream'' operator site

Three sequences are required for complete repression of the aroF promoter by the TyrR repressor protein. Two of these operator sites lie adjacent to each other and overlap the -35 region of the aroF promoter while the third lies about 70 base pairs upstream of the promoter. An aroF-cat (chloramphenicol acetyltransferase) gene fusion has been used to assay the effect of DNA insertions that alter the distance between the two promoter-proximal and the third, distal, operator sites on the repression of the aroF promoter in vivo. The distal site contributes to the repression of the promoter up to a distance of about 400 base pairs and its effect is not dependent on its separation from the first and second sites by an integral number of turns of the DNA helix.