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KMID : 0613820090190030305
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Journal of Life Science
2009 Volume.19 No. 3 p.305 ~ p.310
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Primer RNA Synthesis by E. coli RNA Polymerase on the SSB-coated 229-nt ssi Signal of Lactococcal Plasmid pGKV21
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Jeong Jin-Yong
Kim Sam-Woong
Bahk Jeong-Dong
Kang Ho-Young
Kim Eun-Sil
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Abstract
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Plasmid pGKV21 contains a 229-nucleotide (nt) single-strand DNA initiation (ssi) signal. Using asymmetric PCR, we prepared a small single-stranded (ss) DNA fragment of the ssi signal and, using the 229-nt ssDNA fragment, determined the requirements of RNA polymerase for priming and DNA-protein interaction. The ssi fragment prepared was able to generate primer RNAs with almost the same efficiency as the M13Δlac182/229 phage DNA. However, the cssi (complementary strand of the ssi signal) fragment could not synthesize primer RNAs. This result suggests that the 229-nt ssi signal functions in a strand specific manner. Gel retardation and DNase I footprinting demonstrated that the synthesized ssi fragment could interact with both E. coli RNA polymerase and SSB protein to synthesize primer RNA. In Escherichia coli [pWVAp], an addition of rifampicin resulted in an accumulation of ssDNA, indicating that the host-encoded RNA polymerase is involved in the conversion of ssDNA to double-stranded plasmid DNA.
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KEYWORD
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Primer RNA synthesis, plasmid replication, single-strand initiation DNA(ssi) signal
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