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.     

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.     

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.     

Transcriptional regulation of tyrosine phenol-lyase gene mediated through TyrR and cAMP receptor protein

Using a lac reporter system in Escherichia coli, we showed that the expression of E. herbicola tpl was regulated through TyrR and cAMP receptor protein. Three TyrR boxes upstream of tpl were essential for full expression. The results suggested that the tyrosine-mediated TyrR hexamerization was an important process. The DNA bending between two TyrR boxes, which is triggered by the binding of cAMP receptor protein, may facilitate the conformational change of TyrRs.     

Transcriptional Regulation of Tyrosine Phenol-lyase Gene Mediated through TyrR and cAMP Receptor Protein

Using a lac reporter system in Escherichia coli, we showed that the expression of E. herbicola tpl was regulated through TyrR and cAMP receptor protein. Three TyrR boxes upstream of tpl were essential for full expression. The results suggested that the tyrosine-mediated TyrR hexamerization was an important process. The DNA bending between two TyrR boxes, which is triggered by the binding of cAMP receptor protein, may facilitate the conformational change of TyrRs.     

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

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.     

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.     

Construction of a tyrP-lac operon fusion strain and its use in the isolation and analysis of mutants derepressed for tyrP expression.

The gene tyrP, which codes for a component of the tyrosine-specific transport system, has been localized on the Escherichia coli K-12 chromosome at min 42. A tyrP-lac operon fusion was constructed and used to isolate mutants that have altered expression from the tyrP promoter. All putative tyrP operator mutations were transferred onto a plasmid vector by recombination in vivo. Restriction enzyme analysis of the resultant plasmids suggests that some of these mutants arose from either an insertion or a deletion of DNA occurring within the region of DNA that contains the tyrP promoter.     

Thymine methyls and DNA-protein interactions.

Evidence is summarized showing that thymine methyls are as important in the recognition of specific sequences by proteins as are the more widely recognized hydrogen bonding sites of bases in the major groove (1). Strongest evidence has come from experiments using functional group mutagenesis (2) in which thymines in a specific recognition sequence (e.g., promoters, operators and restriction sites) are replaced by oligonucleotide synthesis with methyl-free uracil or cytosine and 5-methylcytosine. Such experiments have shown that thymine methyls can provide contact points via van der Waals interactions with amino acid side chains of specific DNA binding proteins. Actual contact between a thymine methyl and carbons of a glutamine side chain has been observed in a cocrystal of the phage 434 repressor and its operator by X-ray analysis. The issue of why thymine occurs in DNA is discussed in light of these findings.     

Regulated clustering of variant CD44 proteins increases their hyaluronate binding capacity

Cell contact with the extracellular matrix component hyaluronic acid (HA) plays an important role in many developmental, physiological, and pathological processes, although the regulation of this contact is poorly understood. CD44 proteins carry an amino acid motif that mediates affinity to HA. Artificial clustering of the smallest 85-kD isoform of CD44 (CD44s) has previously been shown to promote binding of the protein to soluble HA (Lesley, J., R. Hyman, and P.W. Kincade. 1993. Adv. Immunol. 54:271-335; Persche, A., J. Lesley, N. English, I. Trowbridge, and R. Hyman. 1995. Eur. J. Immunol. 25:495-501). Here we show that in rat pancreatic carcinoma cells, splice variants of CD44 (CD44v), but not CD44s, form molecular aggregates in the plasma membrane. We demonstrate that reduction-sensitive dimerization of CD44v occurs, and also that larger aggregations of the protein can be stabilized by chemical cross-linking. Different CD44v proteins present on the same cell exclusively form homoaggregates. Molecular clustering does not require an intact cytoplasmic domain of the protein. The ability of cells to bind to soluble HA is upregulated more than one magnitude by the ectopic expression of CD44v4-v7, but only when the CD44v4-v7 protein forms intermolecular aggregates. Tunicamycin treatment inhibits HA binding by CD44v and at the same time destroys oligomerization. We propose that the regulation of clustering of CD44, mediated by factors including the presence of variant exons and glycosylation, allows cells in turn to regulate their HA binding properties.     

Purification of the Escherichia coli purine regulon repressor and identification of corepressors.

The Escherichia coli pur regulon repressor protein was overproduced in a phage T7 expression system. The overexpressed repressor constituted approximately 35% of the soluble cellular protein. Pur repressor was purified to near homogeneity by two chromatographic steps. Hypoxanthine or guanine was required for binding of purified repressor to purF operator DNA. Apparent dissociation constants of 3.4 nM were determined for binding of holorepressor to purF operator and of 1.7 and 7.1 microM were determined for aporepressor interaction with guanine and hypoxanthine, respectively. A requirement for hypoxanthine or guanine for conversion of aporepressor to holorepressor in vitro supports the earlier report (U. Houlberg and K.F. Jensen, J. Bacteriol. 153:837-845, 1983) that these purine bases are involved in regulation of pur gene expression in Salmonella typhimurium and confirms that hypoxanthine and guanine are corepressors.