Cui J, Somerville R L
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153.
J Biol Chem. 1993 Mar 5;268(7):5040-7.
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.
大肠杆菌K12的TyrR蛋白是一种同型二聚体，每个亚基含有513个氨基酸。该蛋白在几个基因的转录调控中起重要作用，这些基因的蛋白质产物催化芳香族氨基酸生物合成或转运过程中的步骤。已开发出有效纯化TyrR蛋白至表观均一性的方法。我们分析了在不存在配体或存在饱和水平的L-酪氨酸、ATP或聚（dI-dC）的情况下，胰蛋白酶对TyrR蛋白的切割模式。在低胰蛋白酶水平（重量比为1:200）且不存在配体时，积累了两种主要的消化产物。这些是22 kDa和31 kDa的多肽，分别显示含有氨基酸残基1-190和191-467。胰蛋白酶的切割模式不受酪氨酸影响。在ATP存在下，观察到一种53 kDa的中间产物，可能含有氨基酸残基1-467。53 kDa产物出现的动力学与ATP在加速R467-F468肽键水解中的作用一致。53 kDa的多肽进一步被胰蛋白酶水解，产生22 kDa和31 kDa的片段。当酪氨酸和ATP都存在时，22 kDa和31 kDa片段的形成速率比不存在这些配体时更快。似乎当两种配体都结合时，R190-Q191和R467-F468肽键的水解速率都增加。额外的有限蛋白酶解实验表明，多肽片段191-467含有ATP结合位点，并且当TyrR蛋白与靶DNA的类似物聚（dI-dC）相互作用时，R190-Q191和R467-F468肽键的切割速率会改变。我们的结果揭示了TyrR蛋白内存在两个主要的结构域。第一个结构域（氨基酸残基1-190）对胰蛋白酶水解具有极强的抗性。第二个结构域（残基191-467）可能含有ATP结合位点，与其他几个对RNA聚合酶的σ54形式响应的启动子特异性转录激活因子同源。TyrR蛋白的其余部分（残基468-513）包含操纵子识别元件，可能以螺旋-转角-螺旋基序的形式排列。该多肽片段未被检测为离散的胰蛋白酶水解产物。
