Reverse transcription of the Mauriceville plasmid of Neurospora. Lack of ribonuclease H activity associated with the reverse transcriptase and possible use of mitochondrial ribonuclease H.
The Mauriceville mitochondrial plasmid of Neurospora encodes a reverse transcriptase that synthesizes a full-length cDNA copy of the major plasmid transcript beginning directly opposite the 3' end of the template RNA (Kuiper, M. T. R., and Lambowitz, A. M. (1988) Cell 55, 693-704). Here, we show that the Mauriceville plasmid reverse transcriptase has no detectable RNase H activity and that cDNAs synthesized either by the column-purified reverse transcriptase or by the endogenous reverse transcriptase in purified ribonucleoprotein particles remain in a full-length duplex with the template RNA. The column-purified Mauriceville plasmid reverse transcriptase initiates cDNA synthesis by using short DNA primers, which remain attached to the 5' end of the (-) strand DNA (Wang, H., Kennell, J. C., Kuiper, M. T. R., Sabourin, J. R., Saldanha, R., and Lambowitz, A. M. (1992) Mol. Cell. Biol. 12, 5131-5144). We find that these primer DNAs can be precisely removed by S1 nuclease digestion of the initial cDNA.RNA duplex, suggesting a mechanism whereby this structure may contribute to primer removal in vivo. Finally, we show that Neurospora mitochondria contain an endogenous RNase H activity, which is present in mitochondrial ribonucleoprotein particle preparations prior to their purification. This mitochondrial RNase H can degrade the endogenous plasmid transcript in ribonucleoprotein particles in vitro and could play a similar role in vivo. The finding that the Mauriceville plasmid reverse transcriptase, which is believed to be a primitive enzyme, has no detectable RNase H activity is consistent with the hypothesis that retroviral reverse transcriptases acquired their RNase H domains from a gene encoding a cellular RNase H.
Wang
H
H
Departments of Molecular Genetics and Biochemistry, Ohio State University, Columbus 43210-1292.
Lambowitz
A M
AM
D000595:Amino Acid Sequence; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D009491:Neurospora / Q000201:enzymology / Q000235:genetics*; D010957:Plasmids; D012331:RNA, Fungal / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016914:Ribonuclease H / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013698:Templates, Genetic
D012331:RNA, Fungal; D012261:Ribonucleoproteins; D008345:Manganese; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H; D015719:Single-Strand Specific DNA and RNA Endonucleases; D008274:Magnesium