FOLDING THE RIBONUCLEASE-H DOMAIN OF MOLONEY MURINE LEUKEMIA-VIRUS REVERSE-TRANSCRIPTASE REQUIRES METAL-BINDING OR A SHORT N-TERMINAL EXTENSION

Reverse transcriptase (RT) is a modular enzyme carrying polymerase and ribonuclease H (RNase H) activities in separable domains. Retroviral replication requires both of these activities. The RNase H domain is r esponsible for hydrolysis of the RNA portion of RNA . DNA hybrids, and this activity requires the presence of divalent cations (Mg2+ or Mn2) that bind its active site. This domain is a part of a large family o f homologous RNase H enzymes of which the RNase HI protein from Escher ichia coli is the best characterized. Although the isolated RNase H do main from human immunodeficiency virus RT is inactive, the Moloney mur ine leukemia virus (MMLV) domain is active in the absence of the polym erase domain, making functional studies more accessible. Using circula r dichroism spectroscopy, we characterized the stability and folding o f two different fragments of MMLV RT that retain RNase H activity. The smaller fragment corresponding to the 157 C-terminal residues of RT i s predominantly unfolded in the absence of divalent cations, but foldi ng can be induced by the addition of metal. The larger fragment corres ponding to the 175 C-terminal residues, however, is stably folded in t he absence of metal. Thus, an 18 residue N-terminal extension outside the region homologous to E. coli RNase HI is important for the structu ral stability of the RNase H domain of MMLV RT, Therefore, this region should be considered part of the RNase H domain. (C) 1998 Wiley-Liss, Inc.