IMPORTANCE OF THE C-TERMINAL HELIX TO THE STABILITY AND ENZYMATIC-ACTIVITY OF ESCHERICHIA-COLI RIBONUCLEASE-H

The ribonuclease H (RNase H) family of enzymes selectively degrades th e RNA strand of RNA DNA hybrids. This activity is essential for retrov iruses such as HIV and resides in a domain of the larger reverse trans criptase molecule. RNase H from Escherichia coli is the best-character ized member of the family and serves as a model for structure/function studies. Despite having almost identical alpha + beta folds, the isol ated domain from HIV is inactive and much less stable than the E. coli homolog. The HIV domain also shows increased disorder in its C-termin al regions (E-helix and His-containing loop). We investigated the impo rtance of this region by studying a variant of E. coli RNase H lacking these elements (RNH Delta E). Despite the elimination of 33 of 155 re sidues (including a complete helix), this C-terminal deletion mutant f olds cooperatively as a subdomain. Surprisingly, this protein lacking residues near the active site retains weak Mn2+-dependent activity. A peptide corresponding to the deleted E-helix is helical in isolation a nd stimulates the activity of the deletion mutant in vitro. These resu lts have implications for the catalytic mechanism of RNase H and drug design targeted to HIV reverse transcriptase.