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 responsible for hydrolysis of the RNA portion of RNA x 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 of homologous RNase H enzymes of which the RNase HI protein from Escherichia coli is the best characterized. Although the isolated RNase H domain from human immunodeficiency virus RT is inactive, the Moloney murine leukemia virus (MMLV) domain is active in the absence of the polymerase domain, making functional studies more accessible. Using circular dichroism spectroscopy, we characterized the stability and folding of two different fragments of MMLV RT that retain RNase H activity. The smaller fragment corresponding to the 157 C-terminal residues of RT is predominantly unfolded in the absence of divalent cations, but folding can be induced by the addition of metal. The larger fragment corresponding to the 175 C-terminal residues, however, is stably folded in the absence of metal. Thus, an 18 residue N-terminal extension outside the region homologous to E. coli RNase HI is important for the structural stability of the RNase H domain of MMLV RT. Therefore, this region should be considered part of the RNase H domain.
Goedken
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ER
Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
Marqusee
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D000595:Amino Acid Sequence; D000818:Animals; D008345:Manganese / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D011487:Protein Conformation; D017510:Protein Folding; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012191:Retroviridae Proteins / Q000737:chemistry* / Q000378:metabolism; D016914:Ribonuclease H / Q000737:chemistry* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D012191:Retroviridae Proteins; D008345:Manganese; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H