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. 1991 Feb;173(3):1208–1214. doi: 10.1128/jb.173.3.1208-1214.1991

Transcription and initiation of ColE1 DNA replication in Escherichia coli K-12.

N Inoue 1, H Uchida 1
PMCID: PMC207244  PMID: 1846858

Abstract

By S1 nuclease protection mapping, we characterized RNA transcripts and nascent ColE1 DNA synthesized in wild-type Escherichia coli cells after infection with lambda-mini-ColE1 hybrid bacteriophages. Transcription of the RNA II region of ColE1 DNA in vivo starts mostly from the RNA II promoter, which was identified by in vitro experiments, and ends at or near the ori site. Synthesis of the leading strand of ColE1 DNA was found to start at the ori site. Nevertheless, the molar ratio of the nascent DNA to the synthesized transcripts ending at the ori site was less than 0.05. In bacterial rnh mutants whose RNase H activities were less than 0.06% of that of the wild type, transcription patterns, as well as nascent DNA synthesis, were still similar to those in rnh+ cells. However, in bacteria whose rnh gene was interrupted by insertion of a drug resistance gene, the number of transcripts ending at the ori site was much reduced and that of transcripts reading through the ori site was definitely increased relative to that observed in wild-type bacteria. These results suggested that cleavage of the RNA transcript at the ori site in vivo is dependent on RNase H activity, as demonstrated in the in vitro system, but most of the cleaved RNA is unable to prime initiation of ColE1 DNA synthesis efficiently.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  2. Campbell A. The steric effect in lysogenization by bacteriophage lambda. I. Lysogenization of a partially diploid strain of Escherichia coli K-12. Virology. 1965 Nov;27(3):329–339. doi: 10.1016/0042-6822(65)90112-1. [DOI] [PubMed] [Google Scholar]
  3. Dasgupta S., Masukata H., Tomizawa J. Multiple mechanisms for initiation of ColE1 DNA replication: DNA synthesis in the presence and absence of ribonuclease H. Cell. 1987 Dec 24;51(6):1113–1122. doi: 10.1016/0092-8674(87)90597-6. [DOI] [PubMed] [Google Scholar]
  4. Dunn J. J., Studier F. W. T7 early RNAs and Escherichia coli ribosomal RNAs are cut from large precursor RNAs in vivo by ribonuclease 3. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3296–3300. doi: 10.1073/pnas.70.12.3296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gesteland R. F. Isolation and characterization of ribonuclease I mutants of Escherichia coli. J Mol Biol. 1966 Mar;16(1):67–84. doi: 10.1016/s0022-2836(66)80263-2. [DOI] [PubMed] [Google Scholar]
  6. Ginsburg D., Steitz J. A. The 30 S ribosomal precursor RNA from Escherichia coli. A primary transcript containing 23 S, 16 S, and 5 S sequences. J Biol Chem. 1975 Jul 25;250(14):5647–5654. [PubMed] [Google Scholar]
  7. Gronenborn B., Messing J. Methylation of single-stranded DNA in vitro introduces new restriction endonuclease cleavage sites. Nature. 1978 Mar 23;272(5651):375–377. doi: 10.1038/272375a0. [DOI] [PubMed] [Google Scholar]
  8. Guarneros G., Montañez C., Hernandez T., Court D. Posttranscriptional control of bacteriophage lambda gene expression from a site distal to the gene. Proc Natl Acad Sci U S A. 1982 Jan;79(2):238–242. doi: 10.1073/pnas.79.2.238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Itaya M. Isolation and characterization of a second RNase H (RNase HII) of Escherichia coli K-12 encoded by the rnhB gene. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8587–8591. doi: 10.1073/pnas.87.21.8587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Itoh T., Tomizawa J. Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H. Proc Natl Acad Sci U S A. 1980 May;77(5):2450–2454. doi: 10.1073/pnas.77.5.2450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Itoh T., Tomizawa J. Initiation of replication of plasmid ColE1 DNA by RNA polymerase, ribonuclease H, and DNA polymerase I. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):409–417. doi: 10.1101/sqb.1979.043.01.047. [DOI] [PubMed] [Google Scholar]
  12. JACOB F., CAMPBELL A. Sur le système de répression assurant l'immunité chez les bactéries lysogenes. C R Hebd Seances Acad Sci. 1959 Jun 1;248(22):3219–3221. [PubMed] [Google Scholar]
  13. Kanaya S., Crouch R. J. The rnh gene is essential for growth of Escherichia coli. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3447–3451. doi: 10.1073/pnas.81.11.3447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kindler P., Keil T. U., Hofschneider P. H. Isolation and characterization of a ribonuclease 3 deficient mutant of Escherichia coli. Mol Gen Genet. 1973 Oct 16;126(1):53–59. doi: 10.1007/BF00333481. [DOI] [PubMed] [Google Scholar]
  15. Kogoma T. Absence of RNase H allows replication of pBR322 in Escherichia coli mutants lacking DNA polymerase I. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7845–7849. doi: 10.1073/pnas.81.24.7845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Messing J., Gronenborn B., Müller-Hill B., Hans Hopschneider P. Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3642–3646. doi: 10.1073/pnas.74.9.3642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  18. Murray N. E., Murray K. Manipulation of restriction targets in phage lambda to form receptor chromosomes for DNA fragments. Nature. 1974 Oct 11;251(5475):476–481. doi: 10.1038/251476a0. [DOI] [PubMed] [Google Scholar]
  19. Naito S., Kitani T., Ogawa T., Okazaki T., Uchida H. Escherichia coli mutants suppressing replication-defective mutations of the ColE1 plasmid. Proc Natl Acad Sci U S A. 1984 Jan;81(2):550–554. doi: 10.1073/pnas.81.2.550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Naito S., Uchida H. Initiation of DNA replication in a ColE1-type plasmid: isolation of mutations in the ori region. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6744–6748. doi: 10.1073/pnas.77.11.6744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Naito S., Uchida H. RNase H and replication of ColE1 DNA in Escherichia coli. J Bacteriol. 1986 Apr;166(1):143–147. doi: 10.1128/jb.166.1.143-147.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ogawa T., Okazaki T. Function of RNase H in DNA replication revealed by RNase H defective mutants of Escherichia coli. Mol Gen Genet. 1984;193(2):231–237. doi: 10.1007/BF00330673. [DOI] [PubMed] [Google Scholar]
  23. Oka A., Nomura N., Morita M., Sugisaki H., Sugimoto K., Takanami M. Nucleotide sequence of small ColE1 derivatives: structure of the regions essential for autonomous replication and colicin E1 immunity. Mol Gen Genet. 1979 May 4;172(2):151–159. doi: 10.1007/BF00268276. [DOI] [PubMed] [Google Scholar]
  24. Oppenheim A., Oppenheim A. B. Regulation of the int gene of bacteriophage lambda: activation by the cII and cIII gene products and the role of the Pi and Pl promoters. Mol Gen Genet. 1978 Sep 20;165(1):39–46. doi: 10.1007/BF00270374. [DOI] [PubMed] [Google Scholar]
  25. Tomizawa J., Masukata H. Factor-independent termination of transcription in a stretch of deoxyadenosine residues in the template DNA. Cell. 1987 Nov 20;51(4):623–630. doi: 10.1016/0092-8674(87)90131-0. [DOI] [PubMed] [Google Scholar]

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