Reverse transcriptase from Escherichia coli exists as a complex with msDNA and is able to synthesize double-stranded DNA.
Reverse transcriptase required for the synthesis of msDNA.Ec67 in an Escherichia coli strain was purified as a large molecular weight complex with msDNA. The complex sedimented in a glycerol gradient at an s value greater than 19. The predominant protein species co-purifying with reverse transcriptase activity in the complex had a molecular weight estimated at 65,000 which is close to the expected size of 67,227 for the Ec67-reverse transcriptase. In addition, the large complex also contained msDNA.Ec67. The purified complex was able to synthesize cDNA using 5 S rRNA as a template (annealed to a synthetic DNA primer), and a double-stranded DNA using a synthetic DNA template (annealed to a synthetic DNA primer). When msDNA.Ec67 was used as a natural template:primer, the purified complex produced two major products: a 103-base single-stranded DNA by extending the 3' end of msDNA using msdRNA as a template, and a 60-base double-stranded DNA product resulting from the converse reaction in which the 3' end of msdRNA is extended using msDNA as a template. The results suggest that bacterial reverse transcriptase is capable of producing single-stranded cDNA and possibly double-stranded DNA as well. Possible implications of these findings on the biology of the msDNA-retron system are discussed.
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854-5635.
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Lampson
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Viswanathan
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Inouye
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Inouye
D001483:Base Sequence; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004277:DNA, Single-Stranded / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes; D010957:Plasmids; D012329:RNA, Bacterial / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D012341:RNA, Ribosomal, 5S / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D015183:Restriction Mapping; D013698:Templates, Genetic
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D012329:RNA, Bacterial; D012341:RNA, Ribosomal, 5S; C058681:msDNA; D012194:RNA-Directed DNA Polymerase