This work is aimed at understanding the molecular basis for specific recognition of nucleic acids by proteins, and the factors involved in their regulatory interactions, both in vivo and in vitro. The work described in this grant proposal focuses on the trp repressor of E. coli (TrpR), with the following goals: 1) Determination of the sequence requirements for tandem, cooperative binding of repressor to DNA; 2) Determination of the binding mode used by the repressor in vivo on natural operators; 3) Measurement of the bending of tandem and single operator DNA's in vitro in the presence and absence of repressor; 4) Measurement of the thermodynamic parameters associated with DNA binding in the tandem and single modes; 5) Determination of the effect of crystallization conditions on specific and nonspecific DNA binding affinity; and 6) Determination of the folding and ligand-binding properties of a fragment of TrpR. %%% To carry out their regulatory functions, proteins exercise precise recognition of nucleic acid sequences, correctly identifying their true targets against a vast background of nonspecific sequences. In the last quarter century, biochemical and genetic studies have pointed to a high degree of chemical complementarily between the protein and nucleic acid partners in specific complexes. The current state of knowledge about the TrpR-DNA interaction suggests that the distinction between specific and non specific binding is due to an interplay of factors, including subtle energetic considerations. The experiments proposed here are aimed at provided a complete picture for TrpR.
