RNase H domain of Moloney murine leukemia virus reverse transcriptase retains activity but requires the polymerase domain for specificity.
The reverse transcriptase-associated RNase H activity of Moloney murine leukemia virus specifically cleaves within the polypurine tract region of the viral genome to generate the primer for plus-strand DNA synthesis and removes the tRNA primer after minus-strand initiation by preferentially cleaving the RNA one nucleotide before the RNA-DNA junction. Moreover, the enzyme is unable to cleave the extended tRNA substrate at the RNA-DNA junction even at high enzyme concentrations. The RNase H domain of the reverse transcriptase was expressed as a glutathione S-transferase fusion protein and purified from Escherichia coli extracts. Following removal of the glutathione S-transferase portion of the protein, the specificity of the isolated RNase H domain was determined in the plus-strand primer reaction and in the tRNA primer removal reaction. Although the isolated domain lacked specificity in both cases, it was still unable to cleave the tRNA substrate precisely at the RNA-DNA junction. Specificity in both cases could be restored by adding back a truncated form of Moloney murine leukemia virus reverse transcriptase lacking the RNase H domain. These results implicate the polymerase domain as a specificity determinant for the RNase H activity of reverse transcriptase. The isolated RNase H domain had higher activity in the presence of Mn2+ than in the presence of Mg2+, but neither the RNase H domain alone nor the RNase H domain coupled to the polymerase domain in wild-type protein exhibited the normal cleavage specificities in the presence of the nonphysiological divalent cation.
Schultz
S J
SJ
Department of Microbiology, School of Medicine, University of Washington, Seattle 98195-7242, USA.
Champoux
J J
JJ
D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005982:Glutathione Transferase; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D012313:RNA; D012343:RNA, Transfer / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D012191:Retroviridae Proteins / Q000235:genetics / Q000378:metabolism*; D016914:Ribonuclease H / Q000235:genetics / Q000378:metabolism*
C034477:RNA primers; D012191:Retroviridae Proteins; D012313:RNA; D012343:RNA, Transfer; D005982:Glutathione Transferase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H