PMID:9188727
| Citation |
Goedken, ER, Raschke, TM and Marqusee, S (1997) Importance of the C-terminal helix to the stability and enzymatic activity of Escherichia coli ribonuclease H. Biochemistry 36:7256-63 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1021/bi970060q |
| Keywords |
Amino Acid Sequence; Circular Dichroism; Crystallography, X-Ray; Enzyme Stability; Escherichia coli/enzymology; Manganese/metabolism; Models, Molecular; Molecular Sequence Data; Protein Conformation; Protein Folding; Protein Structure, Secondary; Ribonuclease H/chemistry; Ribonuclease H/genetics; Ribonuclease H/metabolism; Sequence Deletion |
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