Further genetic analysis of the activation function of the TyrR regulatory protein of Escherichia coli.
Previous reports (J. Cui and R. L. Somerville, J. Bacteriol. 175:1777-1784, 1993; J. Yang, H. Camakaris, and A. J. Pittard, J. Bacteriol. 175:6372-6375, 1993) have identified a number of amino acids in the N-terminal domain of the TyrR protein which are critical for activation of gene expression but which play no role in TyrR-mediated repression. These amino acids were clustered in a single region involving positions 2, 3, 5, 7, 9, 10, and 16. Using random and site-directed mutagenesis, we have identified an additional eight key amino acids whose substitution results in significant or total loss of activator function. All of these are located in the N-terminal domain of TyrR. Alanine scanning at these eight new positions and at five of the previously identified positions for which alanine substitutions had not been obtained has identified three amino acids whose side chains are critical for activation, namely, D-9, R-10, and D-103. Glycine at position 37 is also of critical importance. Alanine substitutions at four other positions (C-7, E-16, D-19, and V-93) caused partial but significant loss of activation, indicating that the side chains of these amino acids also play a contributing role in the activation process.
Yang
J
J
Department of Microbiology, University of Melbourne, Parkville, Victoria, Australia.
Camakaris
H
H
Pittard
A J
AJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000235:genetics*; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000235:genetics*; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C069256:TyrR protein, E coli; D001616:beta-Galactosidase