PMID:8444880
| Citation |
Cui, J and Somerville, RL (1993) The TyrR protein of Escherichia coli, analysis by limited proteolysis of domain structure and ligand-mediated conformational changes. J. Biol. Chem. 268:5040-7 |
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| 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. |
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| Keywords |
Adenosine Triphosphate/metabolism; Amino Acid Sequence; Escherichia coli/chemistry; Escherichia coli Proteins; Hydrolysis; Ligands; Molecular Sequence Data; Peptide Fragments/chemistry; Polydeoxyribonucleotides/metabolism; Protein Conformation; Repressor Proteins/chemistry; Repressor Proteins/metabolism; Transcription Factors/metabolism; Trypsin/metabolism |
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