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http://dx.doi.org/10.5352/JLS.2009.19.3.305

Primer RNA Synthesis by E. coli RNA Polymerase on the SSB-coated 229-nt ssi Signal of Lactococcal Plasmid pGKV21  

Jeong, Jin-Yong (Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine)
Kim, Eun-Sil (Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine)
Kim, Sam-Woong (Division of Biological Sciences, Pusan National University)
Kang, Ho-Young (Division of Biological Sciences, Pusan National University)
Bahk, Jeong-Dong (Division of Applied Life Sciences, Graduate School of Gyeongsang National University)
Publication Information
Journal of Life Science / v.19, no.3, 2009 , pp. 305-310 More about this Journal
Abstract
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 $M13{\Delta}lac182/229$ 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.
Keywords
Primer RNA synthesis; plasmid replication; single-strand initiation DNA (ssi) signal;
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