Structural and functional analysis of the TyrR protein of Escherichia coli

Abstract

The tyrR gene was cloned and precisely located at 29.85 min on the corrected physical map of E. coli. Methods were developed for efficiently purifying the TyrR protein to apparent homogeneity. The pattern of cleavage of the TyrR protein by trypsin, either in the absence of ligands, or in the presence of saturating levels of L-tyrosine, ATP, or poly(dI-dC) was analyzed. At low (1:200 ratio by weight) trypsin levels, in the absence of ligands, two major digestion products accumulated. These were polypeptides of 22 kD and 31 kD. The pattern of trypsin cleavage was unaffected by tyrosine. When both tyrosine and ATP were present, the rate of formation of a 22 kD and a 31 kD fragment was more rapid than in the absence of these ligands. Additional limited proteolysis experiments suggested that polypeptide segment 191-467 contains ATP binding site(s). These results identify two major structural domains with in the TyrR protein. The first domain (amino acid residues 1-190) is extremely resistant to hydrolysis by trypsin. The second domain (residues 191-467), likely to contain ATP binding site(s), is homologous in amino acid sequence to several other transcriptional activators specific for promoters. Furthermore, it was found that the purified TyrR protein has ATPase activity. The functional behavior of the mutant TyrR proteins was evaluated in vivo using single copy lacZ reporter systems based on the mtr promoter or the aroF promoter. A mutant TyrR protein lacking amino acids 2-9 and five additional mutant TyrR proteins were activation-defective. Mutant TyrR proteins lacking amino acid residues 2-9 or 2-19 and three other TyrR mutant proteins retained the ability to repress the aroF promoter, to different extents, in a tyrosine-dependent manner. An additional series of mutant TyrR proteins were constructed and analyzed. In confirmation of previous findings, none of the activation-defective mutant TyrR proteins had lost significant repression function. The TyrR protein was shown by chemical crosslinking to be dimeric. The polypeptide segments critical for dimer formation in vivo were identified by evaluating the negative dominance phenotypes of a series of mutant proteins. Amino acid residues 194-438 were found to contain all the essential dimerization determinants. This segment of the TyrR protein contains an ATP binding site and exhibits a high degree of amino acid sequence homology with the central domains of several transcriptional activators. It is therefore possible that the presence of dimerization determinants is a property that these polypeptide segments have in common. (Abstract shortened by UMI.