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. 2002 Jun;8(6):816–823. doi: 10.1017/s1355838202026730

Probing FinO-FinP RNA interactions by site-directed protein-RNA crosslinking and gelFRET.

Alexandru F Ghetu 1, David C Arthur 1, Tom K Kerppola 1, J N Mark Glover 1
PMCID: PMC1370299  PMID: 12088153

Abstract

The conjugative transfer of F-plasmids is repressed by a two-component system, which consists of the antisense RNA FinP and the protein FinO. FinO binds FinP, protecting it from endonucleolytic degradation and facilitating duplex formation between FinP and its complementary RNA. Here we present the results of site-specific protein-RNA cross-linking and gel-based fluorescence resonance energy transfer (gelFRET) experiments used to probe the structure of a complex of FinO bound to an RNA target consisting of a duplex with 5' and 3' single-stranded tails. The crosslinking experiments reveal that an extensive, largely positively charged surface on FinO contacts RNA. The gelFRET measurements indicate that the 5' single-stranded tail of the RNA is in closer contact with much of the protein than the distal, blunt end of the RNA duplex. These data suggest that significant conformational adjustments in the protein and/or the RNA accompany complex formation.

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Selected References

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  1. Chen Y., Ebright R. H. Phenyl-azide-mediated photocrosslinking analysis of Cro-DNA interaction. J Mol Biol. 1993 Mar 20;230(2):453–460. doi: 10.1006/jmbi.1993.1162. [DOI] [PubMed] [Google Scholar]
  2. Diebold R. J., Rajaram N., Leonard D. A., Kerppola T. K. Molecular basis of cooperative DNA bending and oriented heterodimer binding in the NFAT1-Fos-Jun-ARRE2 complex. Proc Natl Acad Sci U S A. 1998 Jul 7;95(14):7915–7920. doi: 10.1073/pnas.95.14.7915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Finnegan D., Willetts N. The nature of the transfer inhibitor of several F-like plasmids. Mol Gen Genet. 1972;119(1):57–66. doi: 10.1007/BF00270444. [DOI] [PubMed] [Google Scholar]
  4. Frost L. S., Ippen-Ihler K., Skurray R. A. Analysis of the sequence and gene products of the transfer region of the F sex factor. Microbiol Rev. 1994 Jun;58(2):162–210. doi: 10.1128/mr.58.2.162-210.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ghetu A. F., Gubbins M. J., Frost L. S., Glover J. N. Crystal structure of the bacterial conjugation repressor finO. Nat Struct Biol. 2000 Jul;7(7):565–569. doi: 10.1038/76790. [DOI] [PubMed] [Google Scholar]
  6. Ghetu A. F., Gubbins M. J., Oikawa K., Kay C. M., Frost L. S., Glover J. N. The FinO repressor of bacterial conjugation contains two RNA binding regions. Biochemistry. 1999 Oct 19;38(42):14036–14044. doi: 10.1021/bi9911482. [DOI] [PubMed] [Google Scholar]
  7. Hall K. B., Fox R. O. Directed cleavage of RNA with protein-tethered EDTA-Fe. Methods. 1999 May;18(1):78–84. doi: 10.1006/meth.1999.0759. [DOI] [PubMed] [Google Scholar]
  8. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  9. Jerome L. J., Frost L. S. In vitro analysis of the interaction between the FinO protein and FinP antisense RNA of F-like conjugative plasmids. J Biol Chem. 1999 Apr 9;274(15):10356–10362. doi: 10.1074/jbc.274.15.10356. [DOI] [PubMed] [Google Scholar]
  10. Jerome L. J., van Biesen T., Frost L. S. Degradation of FinP antisense RNA from F-like plasmids: the RNA-binding protein, FinO, protects FinP from ribonuclease E. J Mol Biol. 1999 Jan 29;285(4):1457–1473. doi: 10.1006/jmbi.1998.2404. [DOI] [PubMed] [Google Scholar]
  11. Koraimann G., Koraimann C., Koronakis V., Schlager S., Högenauer G. Repression and derepression of conjugation of plasmid R1 by wild-type and mutated finP antisense RNA. Mol Microbiol. 1991 Jan;5(1):77–87. doi: 10.1111/j.1365-2958.1991.tb01828.x. [DOI] [PubMed] [Google Scholar]
  12. Koraimann G., Teferle K., Markolin G., Woger W., Högenauer G. The FinOP repressor system of plasmid R1: analysis of the antisense RNA control of traJ expression and conjugative DNA transfer. Mol Microbiol. 1996 Aug;21(4):811–821. doi: 10.1046/j.1365-2958.1996.361401.x. [DOI] [PubMed] [Google Scholar]
  13. Lee S. H., Frost L. S., Paranchych W. FinOP repression of the F plasmid involves extension of the half-life of FinP antisense RNA by FinO. Mol Gen Genet. 1992 Oct;235(1):131–139. doi: 10.1007/BF00286190. [DOI] [PubMed] [Google Scholar]
  14. Leonard D. A., Kerppola T. K. DNA bending determines Fos-Jun heterodimer orientation. Nat Struct Biol. 1998 Oct;5(10):877–881. doi: 10.1038/2316. [DOI] [PubMed] [Google Scholar]
  15. Mazodier P., Davies J. Gene transfer between distantly related bacteria. Annu Rev Genet. 1991;25:147–171. doi: 10.1146/annurev.ge.25.120191.001051. [DOI] [PubMed] [Google Scholar]
  16. Mullineaux P., Willetts N. Promoters in the transfer region of plasmid F. Basic Life Sci. 1985;30:605–614. doi: 10.1007/978-1-4613-2447-8_42. [DOI] [PubMed] [Google Scholar]
  17. Pendergrast P. S., Chen Y., Ebright Y. W., Ebright R. H. Determination of the orientation of a DNA binding motif in a protein-DNA complex by photocrosslinking. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10287–10291. doi: 10.1073/pnas.89.21.10287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ramirez-Carrozzi V. R., Kerppola T. K. Control of the orientation of Fos-Jun binding and the transcriptional cooperativity of Fos-Jun-NFAT1 complexes. J Biol Chem. 2001 Mar 19;276(24):21797–21808. doi: 10.1074/jbc.M101494200. [DOI] [PubMed] [Google Scholar]
  19. Ramirez-Carrozzi V. R., Kerppola T. K. Dynamics of Fos-Jun-NFAT1 complexes. Proc Natl Acad Sci U S A. 2001 Apr 24;98(9):4893–4898. doi: 10.1073/pnas.091095998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ramirez-Carrozzi V. R., Kerppola T. K. Long-range electrostatic interactions influence the orientation of Fos-Jun binding at AP-1 sites. J Mol Biol. 2001 Jan 19;305(3):411–427. doi: 10.1006/jmbi.2000.4286. [DOI] [PubMed] [Google Scholar]
  21. Ramirez-Carrozzi V., Kerppola T. Gel-based fluorescence resonance energy transfer (gelFRET) analysis of nucleoprotein complex architecture. Methods. 2001 Sep;25(1):31–43. doi: 10.1006/meth.2001.1213. [DOI] [PubMed] [Google Scholar]
  22. Sandercock J. R., Frost L. S. Analysis of the major domains of the F fertility inhibition protein, FinO. Mol Gen Genet. 1998 Oct;259(6):622–629. doi: 10.1007/s004380050856. [DOI] [PubMed] [Google Scholar]
  23. van Biesen T., Frost L. S. The FinO protein of IncF plasmids binds FinP antisense RNA and its target, traJ mRNA, and promotes duplex formation. Mol Microbiol. 1994 Nov;14(3):427–436. doi: 10.1111/j.1365-2958.1994.tb02177.x. [DOI] [PubMed] [Google Scholar]
  24. van Biesen T., Söderbom F., Wagner E. G., Frost L. S. Structural and functional analyses of the FinP antisense RNA regulatory system of the F conjugative plasmid. Mol Microbiol. 1993 Oct;10(1):35–43. doi: 10.1111/j.1365-2958.1993.tb00901.x. [DOI] [PubMed] [Google Scholar]

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