The Mauriceville plasmid of Neurospora crassa: characterization of a novel reverse transcriptase that begins cDNA synthesis at the 3' end of template RNA.
The Mauriceville and Varkud plasmids are retroid elements that propagate in the mitochondria of some Neurospora spp. strains. Previous studies of endogenous reactions in ribonucleoprotein particle preparations suggested that the plasmids use a novel mechanism of reverse transcription that involves synthesis of a full-length minus-strand DNA beginning at the 3' end of the plasmid transcript, which has a 3' tRNA-like structure (M. T. R. Kuiper and A. M. Lambowitz, Cell 55:693-704, 1988). In this study, we developed procedures for releasing the Mauriceville plasmid reverse transcriptase from mitochondrial ribonucleoprotein particles and partially purifying it by heparin-Sepharose chromatography. By using these soluble preparations, we show directly that the Mauriceville plasmid reverse transcriptase synthesizes full-length cDNA copies of in vitro transcripts beginning at the 3' end and has a preference for transcripts having the 3' tRNA-like structure. Further, unlike retroviral reverse transcriptases, the Mauriceville plasmid reverse transcriptase begins cDNA synthesis directly opposite the 3'-terminal nucleotide of the template RNA. The ability to initiate cDNA synthesis directly at the 3' end of template RNAs may also be relevant to the mechanisms of reverse transcription used by LINEs, group II introns, and other non-long terminal repeat retroid elements.
Wang
H
H
Department of Molecular Genetics, Ohio State University, Columbus 43210.
Kennell
J C
JC
Kuiper
M T
MT
Sabourin
J R
JR
Saldanha
R
R
Lambowitz
A M
AM
D001483:Base Sequence; D004271:DNA, Fungal / Q000096:biosynthesis; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000201:enzymology / Q000235:genetics*; D009690:Nucleic Acid Conformation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012331:RNA, Fungal / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D012190:Retroviridae / Q000201:enzymology; D012261:Ribonucleoproteins / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity; D013698:Templates, Genetic
D004271:DNA, Fungal; D012331:RNA, Fungal; D012261:Ribonucleoproteins; D012343:RNA, Transfer; D012194:RNA-Directed DNA Polymerase