Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1987 Dec;169(12):5353–5363. doi: 10.1128/jb.169.12.5353-5363.1987

Transcriptional pausing in a region important for plasmid NR1 replication control.

X N Dong 1, D D Womble 1, R H Rownd 1
PMCID: PMC213958  PMID: 2445727

Abstract

The results of in vitro single-round transcription experiments indicated that RNA polymerase pauses during transcription of the leader region that precedes the repA1 gene of IncFII plasmid NR1. Transcription initiated at either of the two transcription promoter sites of the repA1 gene, which encodes the essential replication initiation protein of NR1, was observed to pause in this region. Pausing was specifically enhanced by addition of NusA protein, an Escherichia coli transcription accessory factor. Northern blot RNA-DNA hybridization analysis of repA1 mRNA synthesized in vivo revealed RNA species that had lengths equivalent to those of the in vitro-paused intermediates. The steady-state rate of in vivo repA1 mRNA transcription downstream from the pause sites (measured by quantitative hybridization of pulse-labeled RNA to DNA probes complementary to different segments of repA1 mRNA) was not appreciably affected, which suggests that the pause sites do not promote premature termination of transcription. The pause sites were located between the target sequence within the leader region of the mRNA that interacts with a 91-base countertranscript and the beginning of the repA1 coding sequence. Because the countertranscript is an inhibitor of translation of repA1 mRNA, transcriptional pausing in this region may be an important feature of the regulation of RepA1 synthesis, which is the mechanism by which plasmid NR1 controls its replication.

Full text

PDF
5353

Images in this article

5356Image
on p.5356
5356Image
on p.5356
5358Image
on p.5358
5358Image
on p.5358

Selected References

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

  1. Adhya S., Gottesman M. Control of transcription termination. Annu Rev Biochem. 1978;47:967–996. doi: 10.1146/annurev.bi.47.070178.004535. [DOI] [PubMed] [Google Scholar]
  2. De Wilde M., Davies J. E., Schmidt F. J. Low molecular weight RNA species encoded by a multiple drug resistance plasmid. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3673–3677. doi: 10.1073/pnas.75.8.3673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dong X., Womble D. D., Luckow V. A., Rownd R. H. Regulation of transcription of the repA1 gene in the replication control region of IncFII plasmid NR1 by gene dosage of the repA2 transcription repressor protein. J Bacteriol. 1985 Feb;161(2):544–551. doi: 10.1128/jb.161.2.544-551.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dretzen G., Bellard M., Sassone-Corsi P., Chambon P. A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal Biochem. 1981 Apr;112(2):295–298. doi: 10.1016/0003-2697(81)90296-7. [DOI] [PubMed] [Google Scholar]
  5. Friedman D. I., Olson E. R. Evidence that a nucleotide sequence, "boxA," is involved in the action of the NusA protein. Cell. 1983 Aug;34(1):143–149. doi: 10.1016/0092-8674(83)90144-7. [DOI] [PubMed] [Google Scholar]
  6. Gerhart E., Wagner H., Nordström K. Structural analysis of an RNA molecule involved in replication control of plasmid R1. Nucleic Acids Res. 1986 Mar 25;14(6):2523–2538. doi: 10.1093/nar/14.6.2523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kassavetis G. A., Chamberlin M. J. Pausing and termination of transcription within the early region of bacteriophage T7 DNA in vitro. J Biol Chem. 1981 Mar 25;256(6):2777–2786. [PubMed] [Google Scholar]
  8. Lacatena R. M., Cesareni G. Base pairing of RNA I with its complementary sequence in the primer precursor inhibits ColE1 replication. Nature. 1981 Dec 17;294(5842):623–626. doi: 10.1038/294623a0. [DOI] [PubMed] [Google Scholar]
  9. Lau L. F., Roberts J. W. Rho-dependent transcription termination at lambda R1 requires upstream sequences. J Biol Chem. 1985 Jan 10;260(1):574–584. [PubMed] [Google Scholar]
  10. Lee F., Yanofsky C. Transcription termination at the trp operon attenuators of Escherichia coli and Salmonella typhimurium: RNA secondary structure and regulation of termination. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4365–4369. doi: 10.1073/pnas.74.10.4365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Light J., Molin S. Post-transcriptional control of expression of the repA gene of plasmid R1 mediated by a small RNA molecule. EMBO J. 1983;2(1):93–98. doi: 10.1002/j.1460-2075.1983.tb01387.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Liu C. P., Churchward G., Caro L. The repA2 gene of the plasmid R100.1 encodes a repressor of plasmid replication. Plasmid. 1983 Sep;10(2):148–155. doi: 10.1016/0147-619x(83)90067-7. [DOI] [PubMed] [Google Scholar]
  13. Luckow V. A., Littlewood R. K., Rownd R. H. Interactive computer programs for the graphic analysis of nucleotide sequence data. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 2):665–673. doi: 10.1093/nar/12.1part2.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Masukata H., Tomizawa J. Control of primer formation for ColE1 plasmid replication: conformational change of the primer transcript. Cell. 1986 Jan 17;44(1):125–136. doi: 10.1016/0092-8674(86)90491-5. [DOI] [PubMed] [Google Scholar]
  15. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  16. Miki T., Easton A. M., Rownd R. H. Cloning of replication, incompatibility, and stability functions of R plasmid NR1. J Bacteriol. 1980 Jan;141(1):87–99. doi: 10.1128/jb.141.1.87-99.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Morita M., Oka A. The structure of a transcriptional unit on colicin E1 plasmid. Eur J Biochem. 1979 Jul;97(2):435–443. doi: 10.1111/j.1432-1033.1979.tb13131.x. [DOI] [PubMed] [Google Scholar]
  18. Morris C. F., Hashimoto H., Mickel S., Rownd R. Round of replication mutant of a drug resistance factor. J Bacteriol. 1974 Jun;118(3):855–866. doi: 10.1128/jb.118.3.855-866.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Olson E. R., Flamm E. L., Friedman D. I. Analysis of nutR: a region of phage lambda required for antitermination of transcription. Cell. 1982 Nov;31(1):61–70. doi: 10.1016/0092-8674(82)90405-6. [DOI] [PubMed] [Google Scholar]
  20. Oxender D. L., Zurawski G., Yanofsky C. Attenuation in the Escherichia coli tryptophan operon: role of RNA secondary structure involving the tryptophan codon region. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5524–5528. doi: 10.1073/pnas.76.11.5524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rosen J., Ryder T., Inokuchi H., Ohtsubo H., Ohtsubo E. Genes and sites involved in replication and incompatibility of an R100 plasmid derivative based on nucleotide sequence analysis. Mol Gen Genet. 1980;179(3):527–537. doi: 10.1007/BF00271742. [DOI] [PubMed] [Google Scholar]
  22. Rosen J., Ryder T., Ohtsubo H., Ohtsubo E. Role of RNA transcripts in replication incompatibility and copy number control in antibiotic resistance plasmid derivatives. Nature. 1981 Apr 30;290(5809):794–797. doi: 10.1038/290794a0. [DOI] [PubMed] [Google Scholar]
  23. Rosenberg M., Court D., Shimatake H., Brady C., Wulff D. L. The relationship between function and DNA sequence in an intercistronic regulatory region in phage lambda. Nature. 1978 Mar 30;272(5652):414–423. doi: 10.1038/272414a0. [DOI] [PubMed] [Google Scholar]
  24. Rownd R. H., Womble D. D., Dong X. N., Luckow V. A., Wu R. P. Incompatibility and IncFII plasmid replication control. Basic Life Sci. 1985;30:335–354. doi: 10.1007/978-1-4613-2447-8_26. [DOI] [PubMed] [Google Scholar]
  25. Rownd R., Nakaya R., Nakamura A. Molecular nature of the drug-resistance factors of the Enterobacteriaceae. J Mol Biol. 1966 Jun;17(2):376–393. doi: 10.1016/s0022-2836(66)80149-3. [DOI] [PubMed] [Google Scholar]
  26. Salser W. Globin mRNA sequences: analysis of base pairing and evolutionary implications. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 2):985–1002. doi: 10.1101/sqb.1978.042.01.099. [DOI] [PubMed] [Google Scholar]
  27. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tamm J., Polisky B. Structural analysis of RNA molecules involved in plasmid copy number control. Nucleic Acids Res. 1983 Sep 24;11(18):6381–6397. doi: 10.1093/nar/11.18.6381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Taylor D. P., Cohen S. N. Structural and functional analysis of cloned DNA segments containing the replication and incompatibility regions of a miniplasmid derived from a copy number mutant of NR1. J Bacteriol. 1979 Jan;137(1):92–104. doi: 10.1128/jb.137.1.92-104.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Thomas P. S. Hybridization of denatured RNA transferred or dotted nitrocellulose paper. Methods Enzymol. 1983;100:255–266. doi: 10.1016/0076-6879(83)00060-9. [DOI] [PubMed] [Google Scholar]
  31. Tomizawa J. Control of ColE1 plasmid replication: the process of binding of RNA I to the primer transcript. Cell. 1984 Oct;38(3):861–870. doi: 10.1016/0092-8674(84)90281-2. [DOI] [PubMed] [Google Scholar]
  32. Tomizawa J., Itoh T. Plasmid ColE1 incompatibility determined by interaction of RNA I with primer transcript. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6096–6100. doi: 10.1073/pnas.78.10.6096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tomizawa J., Itoh T., Selzer G., Som T. Inhibition of ColE1 RNA primer formation by a plasmid-specified small RNA. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1421–1425. doi: 10.1073/pnas.78.3.1421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Winkler M. E., Yanofsky C. Pausing of RNA polymerase during in vitro transcription of the tryptophan operon leader region. Biochemistry. 1981 Jun 23;20(13):3738–3744. doi: 10.1021/bi00516a011. [DOI] [PubMed] [Google Scholar]
  35. Womble D. D., Dong X., Luckow V. A., Wu R. P., Rownd R. H. Analysis of the individual regulatory components of the IncFII plasmid replication control system. J Bacteriol. 1985 Feb;161(2):534–543. doi: 10.1128/jb.161.2.534-543.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Womble D. D., Dong X., Wu R. P., Luckow V. A., Martinez A. F., Rownd R. H. IncFII plasmid incompatibility product and its target are both RNA transcripts. J Bacteriol. 1984 Oct;160(1):28–35. doi: 10.1128/jb.160.1.28-35.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Womble D. D., Rownd R. H. Regulation of IncFII plasmid DNA replication. A quantitative model for control of plasmid NR1 replication in the bacterial cell division cycle. J Mol Biol. 1986 Dec 5;192(3):529–547. doi: 10.1016/0022-2836(86)90274-3. [DOI] [PubMed] [Google Scholar]
  38. Womble D. D., Sampathkumar P., Easton A. M., Luckow V. A., Rownd R. H. Transcription of the replication control region of the IncFII R-plasmid NR1 in vitro and in vivo. J Mol Biol. 1985 Feb 5;181(3):395–410. doi: 10.1016/0022-2836(85)90228-1. [DOI] [PubMed] [Google Scholar]
  39. Womble D. D., Taylor D. P., Rownd R. H. Method for obtaining more-accurate covalently closed circular plasmid-to-chromosome ratios from bacterial lysates by dye-buoyant density centrifugation. J Bacteriol. 1977 Apr;130(1):148–153. doi: 10.1128/jb.130.1.148-153.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wong E. M., Polisky B. Alternative conformations of the ColE1 replication primer modulate its interaction with RNA I. Cell. 1985 Oct;42(3):959–966. doi: 10.1016/0092-8674(85)90292-2. [DOI] [PubMed] [Google Scholar]
  41. Zuker M., Stiegler P. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 1981 Jan 10;9(1):133–148. doi: 10.1093/nar/9.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Zurawski G., Elseviers D., Stauffer G. V., Yanofsky C. Translational control of transcription termination at the attenuator of the Escherichia coli tryptophan operon. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5988–5992. doi: 10.1073/pnas.75.12.5988. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES