In vitro synthesis of multicopy single-stranded DNA, using separate primer and template RNAs, by Escherichia coli reverse transcriptase.
A minor population of wild strains of Escherichia coli contains a retron, a retroelement responsible for the synthesis of multicopy single-stranded DNA (msDNA). The retron is a genetic element consisting of the gene for reverse transcriptase (RT) and the msr-msd region under a single promoter. A single RNA transcript from the msr-msd region serves not only as a template but also as a primer for msDNA synthesis. Here, using a cell-free system with purified RT from retron Ec73, we examined whether the reaction can occur in a bimolecular reaction with use of separately expressed msr and msd transcripts. DNA sequencing of the cell-free product revealed that the sequence of the 5'-end region was identical to that of msDNA-Ec73, indicating that the cDNA synthesis was primed from the 2'-OH group of the specific internal G residue of the primer RNA, identical to the branching G residue in the RNA molecule of msDNA-Ec73. The present results raise an intriguing possibility for a role of bacterial retrons in vivo, the possibility that cellular mRNAs can be converted into cDNAs in retron-harboring cells if the mRNAs contain a sequence complementary to the sequence directly upstream of the branching G residue of the msr RNA transcript.
Shimamoto
T
T
Gene Research Center, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima-naka, Okayama 700, Japan.
Kawanishi
H
H
Tsuchiya
T
T
Inouye
S
S
Inouye
M
M
D004269:DNA, Bacterial / Q000096:biosynthesis; D018076:DNA, Complementary / Q000096:biosynthesis; D004277:DNA, Single-Stranded / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology*; D012313:RNA / Q000378:metabolism*; D012329:RNA, Bacterial / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013698:Templates, Genetic
D004269:DNA, Bacterial; D018076:DNA, Complementary; D004277:DNA, Single-Stranded; C034477:RNA primers; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012313:RNA; D012194:RNA-Directed DNA Polymerase