DIRECT VERSUS INDIRECT READOUT IN THE INTERACTION OF THE TRP REPRESSOR WITH NONCANONICAL BINDING-SITES

Both direct and indirect readouts are utilized when the tup repressor binds to its operators. Here, we use gel-electrophoretic methods to ex amine the role played by direct and indirect readouts in the interacti on 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 c omplexes of the trp repressor with this non-canonical site are lower t han those of the 1:1 complexes formed with either the natural consensu s 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 h igh stability and affinity, similar to that of the 2:1 complex with a consensus sequence found by a selection experiment. The bend angle ind uced on the trpGG target by the binding of one repressor molecule is 2 7 degrees, which is similar to that measured in other 1:1 complexes wi th the repressor. The angle for the 2:1 complex is significantly large r (43 degrees versus 30 degrees in other 2:1 complexes). We present ev idence suggesting that the deleterious effect of the sequence substitu tion in trpGG is compensated by the increased bend angle in the 2:1 co mplex. These observations demonstrate that indirect readout may comple ment for direct readout in determining the nature of the interaction b etween trp repressor and its binding sites. (C) 1998 Academic Press Li mited.