title: Importance of the C-terminal helix to the stability and enzymatic activity of Escherichia coli ribonuclease H.
abstract: The ribonuclease H (RNase H) family of enzymes selectively degrades the RNA strand of RNA-DNA hybrids. This activity is essential for retroviruses such as HIV and resides in a domain of the larger reverse transcriptase molecule. RNase H from Escherichia coli is the best-characterized member of the family and serves as a model for structure/function studies. Despite having almost identical alpha + beta folds, the isolated domain from HIV is inactive and much less stable than the E. coli homolog. The HIV domain also shows increased disorder in its C-terminal regions (E-helix and His-containing loop). We investigated the importance of this region by studying a variant of E. coli RNase H lacking these elements (RNHdeltaE). Despite the elimination of 33 of 155 residues (including a complete helix), this C-terminal deletion mutant folds 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 and stimulates the activity of the deletion mutant in vitro. These results have implications for the catalytic mechanism of RNase H and drug design targeted to HIV reverse transcriptase.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, 94720, USA.
authors.forename: E R
authors.initials: ER
authors.lastname: Goedken
authors.forename: T M
authors.initials: TM
authors.lastname: Raschke
authors.forename: S
authors.initials: S
authors.lastname: Marqusee
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D008345:Manganese; D016914:Ribonuclease H