Direct versus indirect readout in the interaction of the trp repressor with non-canonical binding sites.
Both direct and indirect readouts are utilized when the trp repressor binds to its operators. Here, we use gel-electrophoretic methods to examine the role played by direct and indirect readouts in the interaction of the repressor with a non-canonical binding site, similar to the mtr operator, and named trpGG. The stability and affinity of the 1:1 complexes of the trp repressor with this non-canonical site are lower than those of the 1:1 complexes formed with either the natural consensus sequence or a consensus sequence found in a selection experiment. We attribute this to the inability of the trpGG target to make the same number of water-mediated hydrogen bonds as canonical trp binding sites. On the other hand, the 2:1 complex of the repressor with trpGG has high stability and affinity, similar to that of the 2:1 complex with a consensus sequence found by a selection experiment. The bend angle induced on the trpGG target by the binding of one repressor molecule is 27 degrees, which is similar to that measured in other 1:1 complexes with the repressor. The angle for the 2:1 complex is significantly larger (43 degrees versus 30 degrees in other 2:1 complexes). We present evidence suggesting that the deleterious effect of the sequence substitution in trpGG is compensated by the increased bend angle in the 2:1 complex. These observations demonstrate that indirect readout may complement for direct readout in determining the nature of the interaction between trp repressor and its binding sites.
Bareket-Samish
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Department of Biology, Technion, Technion City, Haifa 32000, Israel.
Cohen
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Haran
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D001426:Bacterial Proteins; D001665:Binding Sites / Q000235:genetics; D016384:Consensus Sequence / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D006860:Hydrogen Bonding; D008968:Molecular Conformation; D015394:Molecular Structure; D009875:Operator Regions, Genetic / Q000235:genetics; D012097:Repressor Proteins / Q000378:metabolism*; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins