SUNY at Albany
June 19-23, 2001
The Role of the Minor Groove at Central Non-Contacted Bases in Operator Recognition by 434 Repressor
The ability of 434 repressor to discriminate between different operators in bacteriophage 434 depends, in part, on the sequence of the central four bases of the operator. Central bases do not make direct contacts with repressor, yet A/T bases at the center of operators have a higher affinity for repressor compared to having G/C at these positions. Previous data from our lab suggests that the ability of the central base pairs to be overtwisted influence sequence dependent differences in affinity.
One hypothesis to explain these observations is that the number of H-bonds in the central region influences the resistance of the operator to twisting flexibility. An alternative model suggests that N2-exocyclic amines act as bulky substituents at the operator center, preventing minor groove compression that occurs in the overtwisted area.
To test these ideas we have created synthetic operators that differ in base composition at the central sites. Non-natural bases were utilized that vary in both the number of hydrogen bonds present and occupancy of an N2 group on purine bases (schematic shown below). Filter binding experiments indicate that repressor affinity for operators is independent of the number of hydrogen bonds present in the central positions. At both central sites, we find that operators lacking an N2-exocyclic amine have at least a two-fold increase in affinity for repressor, regardless of extraneous substituents on the purine base. A difference in the physical properties of DNA in the absence of protein is also noted. A higher Tm (>4.5*C), reduced DNaseI cleavage at the modified central site, and smaller CD transition at ~275nm is always encountered with operators lacking an N2 group protruding in the minor groove of the operator central site. Together the data indicate that the N2-exocyclic amine of central bases affect operator-repressor interactions by modifying intrinsic properties of DNA. Melting temperature data present a model in which the stability of DNA is important in protein affinity (i.e.- those operators that are more stable readily accept the energetic costs associated with binding repressor).
Steven A. Mauro, David R. Pawlowski, Gerald B. Koudelka
Department of Biological Sciences, State University of New York at Buffalo, Buffalo, NY 14260 Fax: (716) 645-2975 e-mail: email@example.com