Dependence of trp repressor-operator affinity, stoichiometry, and apparent cooperativity on DNA sequence and size.
A series of chemically synthesized trp and mutant operator DNAs was employed to examine trp repressor binding. Although only a single repressor-operator complex was observed for most DNAs as reported previously, varying DNA sequence revealed two retarded complexes with an additional band of faster mobility. The relative intensity of the two retarded bands with varying repressor concentrations suggests that cooperative interactions between dimers may occur in the formation of the predominant repressor-operator complex. Direct stoichiometry measurements demonstrated that a 2:1 stoichiometry (two dimers per operator) is found in the primary repressor-operator complex band and that a 1:1 stoichiometry is observed, when present, for the minor repressor-operator complex band of faster mobility. Similar retardation patterns with a single complex of 2:1 stoichiometry were observed for 40-base pair (bp) trp operators corresponding to TrpEDCBA, aroH, and Trp-PL (a derivative of TrpEDCBA with increased symmetry) operator sequences as well as to hybrid operators containing a half-binding site from TrpEDCBA in conjunction with lac operator sequences, although the apparent affinity for the half-site DNAs was diminished by 10-fold. In contrast, the prominence of the 1:1 dimer-operator complex for Trp-PR, a different derivative of TrpEDCBA with increased symmetry, suggests that sequence context may diminish cooperativity between dimers. The stoichiometry observed was also dependent on the length of TrpEDCBA operator DNA used, shifting from primarily 2:1 for 40- and 33-bp TrpEDCBA DNA to primarily 1:1 for 29-, 26-, and 20-bp TrpEDCBA DNAs. In addition, the stability of the repressor-operator complex to electrophoresis is reduced for DNA lengths of 33, 29, and 26 bp. Based on the binding data and footprinting patterns for two hybrid 40-bp TrpEDCBA/lac half-binding site DNAs, it appears that repressor associates tightly to the specific trp half-site, whereas the nonspecific half of the DNA is more loosely bound. These results suggest that repressor dimer-dimer interaction may be an important feature in the trp repressor-operator interaction.
Liu
Y C
YC
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251.
Matthews
K S
KS
D000494:Allosteric Regulation; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008745:Methylation; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D012097:Repressor Proteins / Q000737:chemistry*; D013329:Structure-Activity Relationship; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I; D012097:Repressor Proteins; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins