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.     

A genetic analysis of various functions of the TyrR protein of Escherichia coli.

The TyrR protein is involved in both repression and activation of the genes of the TyrR regulon. Correction of an error in a previously published sequence has revealed a Cro-like helix-turn-helix DNA-binding domain near the carboxyl terminus. Site-directed mutagenesis in this region has generated a number of mutants that can no longer repress or activate. Deletions of amino acid residues 5 to 42 produced a protein that could repress but not activate. The central domain of TyrR contains an ATP-binding site and is homologous with the NtrC family of activator proteins. A mutation to site A of the ATP-binding site and other mutations in this region affect tyrosine-mediated repression but do not prevent activation or phenylalanine-mediated repression of aroG.     

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.     

The predicted protein product of a pathogenicity locus from Pseudomonas syringae pv. phaseolicola is homologous to a highly conserved domain of several procaryotic regulatory proteins.

A ca. 20-kilobase (kb) region (hrp) that controls the interaction of Pseudomonas syringae pv. phaseolicola with its host (pathogenicity) and nonhost plants (hypersensitive reaction) was previously cloned and partially characterized. In this study we defined the limits and determined the nucleotide sequence of a hrp locus (hrpS), located near the right end of the hrp cluster. The largest open reading frame (ORF302) in hrpS has a coding capacity for a 302-amino-acid polypeptide. The predicted amino acid sequence of the translation product of ORF302 (HrpS) shows significant similarity to several procaryotic regulatory proteins, including the NtrC, NifA, and DctD proteins of Rhizobium spp., the NtrC and NifA proteins of Klebsiella pneumoniae, and the TyrR protein of Escherichia coli. These proteins regulate diverse operons involved in nitrogen fixation, transport and metabolism of amino acids, and transport of C-4 dicarboxylic acids. The HrpS protein appears to be the shortest naturally occurring member of this family of proteins, corresponding for the most part to the highly conserved central domain of these proteins, which contains a putative ATP-binding site. A C-terminal segment analogous to the less-well-conserved domain, involved in DNA binding of NtrC and NifA, is also present in HrpS. These similarities suggest that HrpS is a regulatory protein. In line with this prediction is the finding that a functional hrpS gene is necessary for the activation of another hrp locus during the plant-bacterium interaction.     

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

Expression of the mtr gene, which encodes a tryptophan-specific transport system in Escherichia coli K-12, is activated by the TyrR protein. Two TyrR protein binding sites (TYR R boxes) are positioned upstream of the -35 promoter region. Mutational and DNase protection studies indicate that TyrR protein binds preferentially to the TYR R box closest to the promoter, and this is essential for activation of gene expression. In the presence of tyrosine and ATP, a second TyrR molecule is able to cooperatively bind to the second box and cause a further increase in the level of activation.     

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.