• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of narLink to Publisher's site
Nucleic Acids Res. May 25, 1995; 23(10): 1691–1695.
PMCID: PMC306923

RNAII transcribed by IPTG-induced T7 RNA polymerase is non-functional as a replication primer for ColE1-type plasmids in Escherichia coli.

Abstract

RNAII, an RNA species encoded by ColE1-type plasmids, serves as a primer for plasmid DNA replication. Previous work has shown that overproduction of RNAII transcribed by Escherichia coli RNA polymerase results in elevated plasmid copy number. To produce a plasmid in which the elevation of its copy number is inducible, we placed transcription of RNAII under the control of a bacteriophage T7 late promoter regulated by IPTG-inducible T7 RNA polymerase. During induction of T7 RNA polymerase by IPTG, we found that RNAII was overexpressed, but that, surprisingly, this increase in RNAII did not result in elevation of plasmid copy number. These results suggest that RNAII transcribed by T7 RNA polymerase does not function as a primer for plasmid DNA replication. Since RNAII function requires folding of its multiple stem-loop structures in a precise conformation and folding of RNAII can be influenced by its rate of transcription, the extremely rapid rate of travel of the T7 RNA polymerase may preclude proper folding of RNAII during its elongation.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.2M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Masukata H, Tomizawa J. A mechanism of formation of a persistent hybrid between elongating RNA and template DNA. Cell. 1990 Jul 27;62(2):331–338. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Morita M, Oka A. The structure of a transcriptional unit on colicin E1 plasmid. Eur J Biochem. 1979 Jul;97(2):435–443. [PubMed]
  • Cesareni G, Helmer-Citterich M, Castagnoli L. Control of ColE1 plasmid replication by antisense RNA. Trends Genet. 1991 Jul;7(7):230–235. [PubMed]
  • Davison J. Mechanism of control of DNA replication and incompatibility in ColE1-type plasmids--a review. Gene. 1984 Apr;28(1):1–15. [PubMed]
  • Polisky B. ColE1 replication control circuitry: sense from antisense. Cell. 1988 Dec 23;55(6):929–932. [PubMed]
  • Uhlin BE, Schweickart V, Clark AJ. New runaway-replication-plasmid cloning vectors and suppression of runaway replication by novobiocin. Gene. 1983 May-Jun;22(2-3):255–265. [PubMed]
  • Bachvarov D, Jay E, Ivanov I. Construction of a Co1E1 plasmid bearing inducible high-copy-number phenotype. Folia Microbiol (Praha) 1990;35(3):177–182. [PubMed]
  • Golomb M, Chamberlin M. Characterization of T7-specific ribonucleic acid polymerase. IV. Resolution of the major in vitro transcripts by gel electrophoresis. J Biol Chem. 1974 May 10;249(9):2858–2863. [PubMed]
  • Studier FW, Moffatt BA. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. [PubMed]
  • Lin-Chao S, Cohen SN. The rate of processing and degradation of antisense RNAI regulates the replication of ColE1-type plasmids in vivo. Cell. 1991 Jun 28;65(7):1233–1242. [PubMed]
  • Lin-Chao S, Wong TT, McDowall KJ, Cohen SN. Effects of nucleotide sequence on the specificity of rne-dependent and RNase E-mediated cleavages of RNA I encoded by the pBR322 plasmid. J Biol Chem. 1994 Apr 8;269(14):10797–10803. [PubMed]
  • Ghora BK, Apirion D. Structural analysis and in vitro processing to p5 rRNA of a 9S RNA molecule isolated from an rne mutant of E. coli. Cell. 1978 Nov;15(3):1055–1066. [PubMed]
  • Lin-Chao S, Chen WT, Wong TT. High copy number of the pUC plasmid results from a Rom/Rop-suppressible point mutation in RNA II. Mol Microbiol. 1992 Nov;6(22):3385–3393. [PubMed]
  • Lin-Chao S, Bremer H. Effect of the bacterial growth rate on replication control of plasmid pBR322 in Escherichia coli. Mol Gen Genet. 1986 Apr;203(1):143–149. [PubMed]
  • Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, Green MR. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. [PMC free article] [PubMed]
  • Tucker WT, Miller CA, Cohen SN. Structural and functional analysis of the par region of the pSC 10 1 plasmid. Cell. 1984 Aug;38(1):191–201. [PubMed]
  • Cabello F, Timmis K, Cohen SN. Replication control in a composite plasmid constructed by in vitro linkage of two distinct replicons. Nature. 1976 Jan 29;259(5541):285–290. [PubMed]
  • Chamberlin M, Ring J. Characterization of T7-specific ribonucleic acid polymerase. 1. General properties of the enzymatic reaction and the template specificity of the enzyme. J Biol Chem. 1973 Mar 25;248(6):2235–2244. [PubMed]
  • Helmer-Citterich M, Anceschi MM, Banner DW, Cesareni G. Control of ColE1 replication: low affinity specific binding of Rop (Rom) to RNAI and RNAII. EMBO J. 1988 Feb;7(2):557–566. [PMC free article] [PubMed]
  • Lewicki BT, Margus T, Remme J, Nierhaus KH. Coupling of rRNA transcription and ribosomal assembly in vivo. Formation of active ribosomal subunits in Escherichia coli requires transcription of rRNA genes by host RNA polymerase which cannot be replaced by bacteriophage T7 RNA polymerase. J Mol Biol. 1993 Jun 5;231(3):581–593. [PubMed]
  • Lopez PJ, Iost I, Dreyfus M. The use of a tRNA as a transcriptional reporter: the T7 late promoter is extremely efficient in Escherichia coli but its transcripts are poorly expressed. Nucleic Acids Res. 1994 Apr 11;22(7):1186–1193. [PMC free article] [PubMed]
  • Tomizawa JI, Itoh T. The importance of RNA secondary structure in CoIE1 primer formation. Cell. 1982 Dec;31(3 Pt 2):575–583. [PubMed]
  • Rommens J, MacKnight D, Pomeroy-Cloney L, Jay E. Gene expression: chemical synthesis and molecular cloning of a bacteriophage T5 (T5P25) early promoter. Nucleic Acids Res. 1983 Sep 10;11(17):5921–5940. [PMC free article] [PubMed]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links