title: The putative substrate recognition loop of Escherichia coli ribonuclease H is not essential for activity.
abstract: The RNase H family of enzymes catalyzes the hydrolysis of RNA from RNA DNA hybrids in a divalent metal-dependent fashion. To date, structure/function studies have focused on two members of this family: Escherichia coli RNase HI, a small monomeric protein; and human immunodeficiency virus, type I (HIV) RNase H, a domain of HIV reverse transcriptase. The isolated RNase H domain from HIV reverse transcriptase can be expressed independently and shares significant structural homology with its E. coli homologue; however, unlike the bacterial protein, it is inactive. The most notable difference between the inactive domain from HIV and the active E. coli protein is a basic helix/loop sequence, present in E. coli but absent from the HIV homologue. Substitution of this basic region into the HIV domain partially restores its activity and increases its thermodynamic stability. By deleting the basic helix/loop 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 basic helix/loop is not required for RNase H activity.
authors.affiliation: Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Keck
authors.forename: S
authors.initials: S
authors.lastname: Marqusee
mesh_terms: D000595:Amino Acid Sequence; D002413:Cations, Divalent / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006678:HIV / Q000201:enzymology; D018257:Helix-Loop-Helix Motifs; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016914:Ribonuclease H / Q000737:chemistry*; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D002413:Cations, Divalent; D008345:Manganese; D016914:Ribonuclease H