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

Abstract

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.The tyrP gene encodes a cytoplasmic membrane protein which represents the tyrosine-specific transport system of Escherichia coli (28,30). Its expression is repressed when cells are grown in medium supplemented with tyrosine but is activated when phenylalanine but not tyrosine is present in the medium (13, 29). When both tyrosine and phenylalanine are present, repression is dominant. When cells are grown in minimal medium, expression of tyrP is partially repressed. Both repression and activation require functional TyrR protein, making tyrP a member of the TyrR regulon (14, 29).The tyrP gene and its upstream region have been sequenced, and two adjacent TYR R boxes, one of which overlaps the -35 region of a likely promoter, have been identified (13). TYR R boxes are related to the palindromic sequence TGTAAA-N6-TTTACA and, in every case in the TyrR regulon in which repression is mediated by TyrR protein and tyrosine, two adjacent boxes have been found to occur (5,6,9,11,13,22,31).Two mutants in which a base change in the right-hand box of tyrP has resulted in a decrease in tyrosine repression but not in phenylalanine activation have been described (13). In these studies, in which Si nuclease was used to identify the transcription start site in tyrP, the presence of two promoters was reported. One of these was positioned so that its -35 region overlapped the right-hand TYR R box, whereas the other was located some 40 bases further upstream. This latter transcription start site seemed to be specifically activated by growth in Casamino Acids and to a lesser extent by growth in the presence of phenylalanine. Other experiments showed that the Casamino Acids effect was independent of TyrR.In this paper, we report new experiments that establish that there is in fact only one promoter for tyrP, confirm its position overlapping the right-hand TYR R box, and offer an explanation for the apparent presence of a second promoter in previous results. In add...

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

“…This will be discussed separately later. Mutational studies have confirmed the critical role of the 'weak' box in the repression of three of the four transcription units, namely uroF-tyrA, tyrP and tyvB (Cobbett & Delbridge 1987;Yang & Pittard 1987;Andrews et al 1991b). Similar studies have shown that the 'weak' box of avoL is not required for repression (Lawley & Pittard 1994).…”

Molecular analysis of the TyrR protein-mediated activation of mtr gene expression in Escherichia coli K-12

“…It also requires an adjacent strong box. The inactivation of this box abolishes protection of both boxes, except at high concentrations of TyrR protein, and results in the loss of TyrR-mediated regulation of both genes (1,2,16,25). In tyrP, the tyrosine-mediated cooperative binding results in repression, whereas in mtr, because of the different placement of the second TYR R box, it results in a further increase in activation.…”

“…In the case of tyrP, this weak box is separated by 1 bp from the strong box and lies downstream, overlapping the RNA polymerase binding site (1,2). In the case of mtr, it is situated 8 bp upstream of the strong box and well clear of the RNA polymerase binding site.…”

“…In every case the double boxes are separated by a single base pair, and expression from the relevant promoter is repressed by TyrR protein in the presence of tyrosine. DNase I protection studies show that in the absence of tyrosine, TyrR protein protects only one of each of these double boxes (1)(2)(3)28). The boxes protected by TyrR protein alone are referred to as strong boxes, are closely homologous to the TYR R box consensus sequence, and in general lie outside the RNA polymerase binding site (4,22).…”

Evidence for involvement of proteins HU and RpoS in transcription of the osmoresponsive proU operon in Escherichia coli

“…The plasmids pHYD272, pHYD275, pHYD283, and pHYD284 have been described earlier (6) and are a panel ofproU-bearing derivatives of the low-copy-number promotercloning vector pMU575 that contains 1acZ+ as the reporter gene and encodes Tpr as the antibiotic resistance marker (2).…”