Tryptophan super-repressors with alanine 77 changes.
The binding of L-tryptophan to Escherichia coli tryptophan aporepressor enables the holorepressor complex to bind operator DNA tightly. The side chain of residue alanine 77 is located in one of the most flexible regions of Trp repressor, between residues critical for binding DNA. Codon-directed mutagenesis was used to make genes encoding mutant Trp repressors with each of the 19 naturally occurring amino acid changes of Ala77. The 19 mutant proteins are made at the same steady-state levels as wild type. Sensitive challenge phage assays show that 7 of the 19 mutant proteins (Cys, Ser, Val, Leu, Thr, Ile, and Lys) are more active than wild-type protein when tryptophan is limiting in vivo. Among these 7 mutant super-aporepressors, proteins with Cys and Ser changes also are super-holorepressors, because they repress better than wild-type holorepressor when tryptophan is in excess. These results and others suggest that super-aporepressors associate more poorly than wild-type aporepressor with nonspecific DNA. Consistent with this idea, these 7 changes are predicted to disrupt the tertiary structure of aporepressor, but have more limited effects on the structure of holorepressor.
Arvidson
D N
DN
California Institute of Biological Research, La Jolla 92037.
Pfau
J
J
Hatt
J K
JK
Shapiro
M
M
Pecoraro
F S
FS
Youderian
P
P
D000409:Alanine / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001059:Apoproteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins; D001483:Base Sequence; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*
D001059:Apoproteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; C027190:TRPR protein, E coli; D014364:Tryptophan; D000409:Alanine