De novo and DNA primer-mediated initiation of cDNA synthesis by the mauriceville retroplasmid reverse transcriptase involve recognition of a 3' CCA sequence.
The Mauriceville mitochondrial retroplasmid of Neurospora encodes a novel reverse transcriptase that initiates cDNA synthesis at a 3' tRNA-like structure of the plasmid transcript, either de novo (i.e. without a primer) or by using the 3' OH group of a DNA primer. Both the de novo and primer-mediated initiations involve recognition of structural features at the 3' end of the retroplasmid transcript, which ends with a 3' CCACCA. Here, detailed biochemical characterization of the retroplasmid reverse transcriptase shows that the 3' CCA of the plasmid transcript is the major structural feature recognized by the reverse transcriptase for both the de novo and primer-mediated initiations. Complementarity between the DNA primer and RNA template is not required for the primer-mediated initiation, although short (1 to 3 nt) base-pairing interactions can influence both the efficiency and site of initiation near the 3' end of the transcript. Single nucleotide changes in the 3' CCA lead to less efficient initiation in the upstream CCA with an increased propensity to add extra "non-coded" nucleotides to the 5' end of the cDNA during de novo initiation or to the 3' end of the primer during primer-mediated initiation. Secondary structure features upstream of the 3' CCA also influence the efficiency of initiation, but are not stringently required in vitro. Finally, we find that the retroplasmid reverse transcriptase does not efficiently use DNA primers that are base-paired to internal positions in the RNA template, nor does it use analogs of natural substrates used by non-long terminal repeat retrotransposon or retroviral reverse transcriptases. Our results indicate that the retroplasmid reverse transcriptase is uniquely adapted to initiate cDNA synthesis by recognizing a 3' CCA sequence. The ability to recognize a specific template sequence is common for RNA polymerases, but unprecedented for a reverse transcriptase.
Chen
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Department of Molecular Genetics, Ohio State University, 484 West Twelfth Avenue, Columbus, OH, 43210-1292, USA.
Lambowitz
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AM
D001482:Base Composition; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis*; D019143:Evolution, Molecular; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000201:enzymology / Q000235:genetics*; D009690:Nucleic Acid Conformation; D010957:Plasmids; D012331:RNA, Fungal; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D018626:Retroelements; D013698:Templates, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D012331:RNA, Fungal; D018626:Retroelements; D012194:RNA-Directed DNA Polymerase