THE PUTATIVE SUBSTRATE RECOGNITION LOOP OF ESCHERICHIA-COLI RIBONUCLEASE-H IS NOT ESSENTIAL FOR ACTIVITY

The RNase H family of enzymes catalyzes the hydrolysis of RNA from RNA . DNA hybrids in a divalent metal-dependent fashion. To date, structu re/function studies have focused on two members of this family: Escher ichia coli RNase HI, a small monomeric protein; and human immunodefici ency virus, type I (HIV) RNase H, a domain of HIV reverse transcriptas e. The isolated RNase H domain from HIV reverse transcriptase can be e xpressed independently and shares significant structural homology with its E. coli homologue; however, unlike the bacterial protein, it is i nactive. The most notable difference between the inactive domain from HIV and the active E. coli protein is a basic helix/loop sequence, pre sent in E. coli but absent from the HIV homologue. Substitution of thi s basic region into the HIV domain partially restores its activity and increases its thermodynamic stability, By deleting the basic helix/lo op region, we have modeled the structural difference between these two polypeptides onto the E. coli homologue. Surprisingly, the resulting mutant protein is active in Mn2+-dependent fashion. Therefore, the bas ic helix/loop is not required for RNase H activity.