FAST FOLDING MUTANTS OF TETRAHYMENA RIBOZYME

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The three dimensional structure of TBP consists of two nearly identical domains that adopt a saddle shape. The concave undersurface of the C-terminal domain of this saddle-shaped protein consists of highly curved antiparallel ?-sheets which contain all of the amino acids implicated in DNA binding. The convex upper surface of the saddle contains residues important for interactions with other transcription factors. The DNA binding sites consist of two pairs of phenylalanine residues on both sides of the concave saddle. These residues are predicted to be solvent accessible in the unbound monomeric protein. However, whether the non-conserved N-terminal domain interacts with these residues is not known. Similarly, the solvent accessibility of the DNA-binding site of the protein in oligomeric TBP is also not known. Answers to these questions are of fundamental importance to understanding the mechanism of binding and function of this protein. The initial aim of this study is to compare the solvent accessibility of these phenylalanine residues in unbound monomeric TBP, oligomeric TBP and TBP-DNA complexes. Preliminary mass Hspectrometric studies have revealed that the phenylalanine containing Hpeptides are modified in a time scale of 4 msec upon exposure at Hbeamline X9A.