Cloning, expression, and purification of a catalytic fragment of Moloney murine leukemia virus reverse transcriptase: crystallization of nucleic acid complexes.
Reverse transcriptase is an essential retroviral enzyme that uses RNA- and DNA-directed DNA polymerase activities as well as an RNaseH activity to synthesize a double-stranded DNA copy of the single-stranded RNA genome. In an effort to obtain high-resolution structural information regarding the polymerase active site of reverse transcriptase, we have pursued studies on a catalytic fragment from Moloney murine leukemia virus reverse transcriptase. DNA encoding the catalytic fragment, defined originally by limited proteolytic digestion, has been cloned, and the protein has been expressed and purified from Escherichia coli. The fragment obtained by limited proteolytic digestion and the bacterially expressed fragnment retain polymerase activity. Crystallization studies involving nucleic acid complexes with a catalytic fragment from both sources are reported, including variables screened to improve crystals and cryocooling. Three crystal forms of catalytic fragment-nucleic acid complexes have been characterized, which all contain at least two protein molecules in the asymmetric unit. As isolated, the catalytic fragment is monomeric. This analysis indicates that the enzyme dimerizes in the presence of nucleic acid.
Waksman Institute and Department of Chemistry, Rutgers University, Piscataway, New Jersey 08854, USA.
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Sun
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Jessen
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Liu
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Liu
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Najmudin
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Georgiadis
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D003460:Crystallization; D018360:Crystallography, X-Ray; D004247:DNA; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D005615:Freezing; D005976:Glutaral; D008970:Molecular Weight; D008979:Moloney murine leukemia virus / Q000201:enzymology* / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010447:Peptide Hydrolases; D012194:RNA-Directed DNA Polymerase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
D003432:Cross-Linking Reagents; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D010447:Peptide Hydrolases; D005976:Glutaral