Statistical and structural analysis of trp binding sites: comparison of natural and in vitro selected sequences

Two different modes can be used when the trp repressor binds to trp binding sites. In the "full-site mode" each repressor molecule is bound to a DNA target containing at least two conserved five base pair tracts separated by eight base pairs. The binding of the repressor to natural trp operators is of this kind. In the "half-site mode" two repressor molecules are sequence-specifically bound, with infinite cooperativity, to two abutting DNA pentamers. We present evidence suggesting that the sequences obtained by a recent in vitro selection assay (Czernik et al. J. Biol. Chem. 269, 27869-27875, 1994) were selected by the binding of two repressor molecules, and that the repressor is bound to most of these sequences using the half-site mode. Using the results of the selection assay, and the set of natural trp binding sites, we characterize the different sequence requirements of the "full-site" versus the "half-site" binding modes. A statistical analysis of the information content of these binding sites shows that functional information on protein binding modes can be extracted from a set of DNA binding sites by comparing the information content of two different DNA populations, or sub-populations. Furthermore, it shows that the binding of proteins to sequences selected by a functional in vitro assay do not necessarily mimic the binding of the protein to the natural targets, even if the information content is similar in the two DNA target populations, i.e., even if the stringency of the selection assay is adequate for locating natural-like sequences. In addition, we show that the structural requirements for protein-DNA interactions can be achieved by different conformations at the base-pair level. Differences in the structural characteristics of different base-pair steps can be used to determine the binding mode and differential binding affinity, which can be utilized in the regulation of several binding sites by a single specific protein.