The Mauriceville retroplasmid reverse transcriptase initiates cDNA synthesis de novo at the 3' end of tRNAs.
The Mauriceville retroplasmid of Neurospora mitochondria encodes a novel reverse transcriptase that initiates cDNA synthesis de novo (i.e., without a primer) at the 3' CCA of the plasmid transcript's 3' tRNA-like structure (H. Wang and A. M. Lambowitz, Cell 75:1071-1081, 1993). Here, we show that the plasmid reverse transcriptase also initiates cDNA synthesis de novo at the 3' end of tRNAs, leading to synthesis of a full-length cDNA copy of the tRNA. The use of tRNA templates in vivo was suggested previously by the structure of suppressive mutant plasmids that have incorporated mitochondrial tRNA sequences (R. A. Akins, R. L. Kelley, and A. M. Lambowitz, Cell 47:505-516, 1986). The in vitro experiments show that efficient de novo initiation on tRNA templates requires an unpaired 3' CCA and occurs predominantly opposite position C-2 of the 3' CCA sequence, the same position as in the plasmid transcript. In other reactions, the plasmid reverse transcriptase synthesizes cDNA dimers by template switching between two tRNA templates and initiates at an internal position in a tRNA by using the 3' end of the tRNA as a primer. Finally, we show that template switching between the tRNA and the plasmid transcript in vitro gives rise to hybrid cDNAs of the type predicted to be intermediates in the generation of the suppressive mutant plasmids. The ability of the plasmid reverse transcriptase to initiate at the 3' end of tRNAs presumably reflects the recognition of structural features similar to those of the 3' tRNA-like structure of the plasmid transcript. The recognition of tRNAs or tRNA-like structures as templates for cDNA synthesis may be characteristic of primitive reverse transcriptases that evolved from RNA-dependent RNA polymerases.
Chiang
C C
CC
Department of Molecular Genetics, The Ohio State University, Columbus 43210-1292, USA.
Lambowitz
A M
AM
D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis*; D004271:DNA, Fungal / Q000096:biosynthesis*; D008957:Models, Genetic; D009154:Mutation; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D012364:RNA, Transfer, Trp / Q000235:genetics / Q000378:metabolism; D012365:RNA, Transfer, Tyr / Q000235:genetics / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013698:Templates, Genetic
D018076:DNA, Complementary; D004271:DNA, Fungal; D012364:RNA, Transfer, Trp; D012365:RNA, Transfer, Tyr; D012194:RNA-Directed DNA Polymerase