Requirements of the secondary structures in the primary transcript for multicopy single-stranded DNA synthesis by reverse transcriptase from bacterial retron-Ec107.
Multicopy single-stranded DNA (msDNA) is produced by bacterial retroelements called retrons. It consists of single-stranded DNA that is linked to an internal G residue of an RNA molecule by a 2',5'-phosphodiester linkage. It has been demonstrated that specific primary sequences, as well as the secondary structures immediately downstream of the G residue, are essential for the cDNA priming reaction (Shimamoto, T., Hsu, M.-Y., Inouye, S., and Inouye, M. (1993) J. Biol. Chem. 268, 2684-2692). We have now examined the requirement of the structures in the region corresponding to DNA for msDNA synthesis. The upper stem region consisting of 71 bases of msDNA-Ec107 was found not to be essential, and this region could be deleted to efficiently produce a truncated msDNA containing only a 36-base single-stranded DNA. Various mutations including base replacements, deletions, and insertions were constructed in the lower stem region. It was found that any mutations resulting in more stable secondary structures caused reduction in msDNA synthesis. The results indicated that reverse transcriptase requires a loose secondary structure in the template RNA near the cDNA priming site for cDNA elongation.
Shimada
M
M
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854.
Inouye
S
S
Inouye
M
M
D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000096:biosynthesis* / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D017384:Sequence Deletion; D013698:Templates, Genetic; D014158:Transcription, Genetic
D017931:DNA Primers; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D012194:RNA-Directed DNA Polymerase