Abstract
The TyrR protein of Escherichia coli K12 is a homodimer containing 513 amino acids/subunit. This protein is important in the transcriptional regulation of several genes whose protein products catalyze steps in aromatic amino acid biosynthesis or transport. Methods were developed for efficiently purifying the TyrR protein to apparent homogeneity. We analyzed 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). At low (1:200 ratio by weight) trypsin levels, in the absence of ligands, two major digestion products accumulated. These were polypeptides of 22 and 31 kDa, shown to contain amino acid residues 1-190 and 191-467, respectively. The pattern of trypsin cleavage was unaffected by tyrosine. In the presence of ATP, an intermediate species of 53 kDa, probably containing amino acid residues 1-467, was observed. The kinetics of appearance of the 53-kDa species were consistent with a role for ATP in accelerating the hydrolysis of the R467-F468 peptide bond. The 53-kDa polypeptide underwent further tryptic hydrolysis to yield fragments of 22 and 31 kDa. When both tyrosine and ATP were present, the rate of formation of the 22- and 31-kDa fragments was more rapid than in the absence of these ligands. It appears that when both ligands are bound, the rates of hydrolysis of peptide bonds R190-Q191 and R467-F468 are both enhanced. Additional limited proteolysis experiments suggested that polypeptide segment 191-467 contains ATP binding site(s), and that the rate of cleavage of peptide bonds R190-Q191 and R467-F468 is altered when the TyrR protein interacts with poly(dI-dC), an analog of target DNA. Our results reveal the presence of two major structural domains within the TyrR protein. The first domain (amino acid residues 1-190) is extremely resistant to hydrolysis by trypsin. The second domain (residues 191-467), which is likely to contain ATP-binding site(s), is homologous to several other transcriptional activators specific for promoters responsive to the sigma 54 form of RNA polymerase. The remainder of the TyrR protein (residues 468-513) contains the operator recognition elements, probably arranged in the form of a helix-turn-helix motif. This polypeptide segment was not detected as a discrete tryptic hydrolysis product.
Highlights
The TyrR proteinof Escherichia coli K12 isa hom- a homodimer of 513 amino acids/subunit (Fig. 1)
The 53-kDa polypeptide undere- nt, but ATP and either tyrosine or phenylalanine must be went further tryptic hydrolysis toyield fragments of present in order for the TyrR protein to bind to weak boxes
The fragments was more rapid than in the abseonf ctheese mechanisms of repression and activation mediated by the ligands. It appears thatwhen both ligands are bound, TyrR protein have not been established. the rates of hydrolysis of peptide bonds R1s0-Q181and In solution, the TyrRprotein binds ATP with a K d of 9 pM
Summary
504 1 sumably in the form of a helix-turn-helix motif (reviewed by Pittard and Davidson, 1991). Limited trypsin digestion was employed in detecting conformational changeswithin the TyrRprotein in response to thebinding of ligands. Hydroxylapatite (BioGel H T P ) was obtained from BioRad. Chromatographic columns and accessories were purchased from Pharmacia LKB Biotechnology Inc. Buffer A was 10 mM KzHP04/KH2P04 (pH6.6) with 100 mM NaCI, 1 mM EDTA, 0.01% NaN3, 7 mM 8-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride. After 20 min at 37 "C, the phage-cell mixture was transferred into 500 mlofNZC medium (Williams and Blattner, 1979) containing 0.5% maltose, in a 2-liter flask. The supernatant, consisting of a high titer phage lysate, was stored at 0-5 "C prior to use. This procedure reproducibly yielded lysate a t a titer of 2-3 X lO'"/ml
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