Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2004 Jan 1;377(Pt 1):17–23. doi: 10.1042/BJ20030703

Both N- and C-terminal regions are essential for cinnamomin A-chain to deadenylate ribosomal RNA and supercoiled double-stranded DNA.

Wen-Jun He 1, Wang-Yi Liu 1
PMCID: PMC1223831  PMID: 12952522

Abstract

Cinnamomin is a type II ribosome-inactivating protein and its A-chain exhibits RNA N-glycosidase activity to remove an adenine in the conserved sarcin/ricin loop of the largest RNA in ribosome, arresting protein synthesis at the elongation step. In this report, deadenylation of both rRNA and supercoiled DNA by native and recombinant cinnamomin A-chain expressed in Escherichia coli was demonstrated. However, the mutants of cinnamomin A-chain devoid of N-terminal 52 or/and C-terminal 51 amino acid residues lost both the activity of RNA N-glycosidase and the ability to release adenines from supercoiled DNA. Additionally, supercoiled DNA could not be cleaved into nicked and linear forms by these mutants. These results indicate that both N- and C-terminal regions are essential for the cinnamomin A-chain to deadenylate rRNA and supercoiled DNA. It was suggested that phosphodiester bonds in the extensively deadenylated region of supercoiled DNA would become fragile and liable to be broken spontaneously owing to the existence of tension in the supercoiled DNA.

Full Text

The Full Text of this article is available as a PDF (216.8 KB).

Selected References

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

  1. Barbieri L., Battelli M. G., Stirpe F. Ribosome-inactivating proteins from plants. Biochim Biophys Acta. 1993 Dec 21;1154(3-4):237–282. doi: 10.1016/0304-4157(93)90002-6. [DOI] [PubMed] [Google Scholar]
  2. Barbieri L., Gorini P., Valbonesi P., Castiglioni P., Stirpe F. Unexpected activity of saporins. Nature. 1994 Dec 15;372(6507):624–624. doi: 10.1038/372624a0. [DOI] [PubMed] [Google Scholar]
  3. Barbieri L., Valbonesi P., Bonora E., Gorini P., Bolognesi A., Stirpe F. Polynucleotide:adenosine glycosidase activity of ribosome-inactivating proteins: effect on DNA, RNA and poly(A). Nucleic Acids Res. 1997 Feb 1;25(3):518–522. doi: 10.1093/nar/25.3.518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Day P. J., Lord J. M., Roberts L. M. The deoxyribonuclease activity attributed to ribosome-inactivating proteins is due to contamination. Eur J Biochem. 1998 Dec 1;258(2):540–545. doi: 10.1046/j.1432-1327.1998.2580540.x. [DOI] [PubMed] [Google Scholar]
  6. Endo Y., Mitsui K., Motizuki M., Tsurugi K. The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins. J Biol Chem. 1987 Apr 25;262(12):5908–5912. [PubMed] [Google Scholar]
  7. Hosur M. V., Nair B., Satyamurthy P., Misquith S., Surolia A., Kannan K. K. X-ray structure of gelonin at 1.8 A resolution. J Mol Biol. 1995 Jul 14;250(3):368–380. doi: 10.1006/jmbi.1995.0383. [DOI] [PubMed] [Google Scholar]
  8. Huang P. L., Chen H. C., Kung H. F., Huang P. L., Huang P., Huang H. I., Lee-Huang S. Anti-HIV plant proteins catalyze topological changes of DNA into inactive forms. Biofactors. 1992 Dec;4(1):37–41. [PubMed] [Google Scholar]
  9. Kitaoka Y. Involvement of the amino acids outside the active-site cleft in the catalysis of ricin A chain. Eur J Biochem. 1998 Oct 1;257(1):255–262. doi: 10.1046/j.1432-1327.1998.2570255.x. [DOI] [PubMed] [Google Scholar]
  10. Li M. X., Yeung H. W., Pan L. P., Chan S. I. Trichosanthin, a potent HIV-1 inhibitor, can cleave supercoiled DNA in vitro. Nucleic Acids Res. 1991 Nov 25;19(22):6309–6312. doi: 10.1093/nar/19.22.6309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lindahl T., Andersson A. Rate of chain breakage at apurinic sites in double-stranded deoxyribonucleic acid. Biochemistry. 1972 Sep 12;11(19):3618–3623. doi: 10.1021/bi00769a019. [DOI] [PubMed] [Google Scholar]
  12. Ling J., Liu W. Y., Wang T. P. Cleavage of supercoiled double-stranded DNA by several ribosome-inactivating proteins in vitro. FEBS Lett. 1994 May 30;345(2-3):143–146. doi: 10.1016/0014-5793(94)00421-8. [DOI] [PubMed] [Google Scholar]
  13. Ling J., Liu W. Y., Wang T. P. Simultaneous existence of two types of ribosome-inactivating proteins in the seeds of Cinnamonum camphora--characterization of the enzymatic activities of these cytotoxic proteins. Biochim Biophys Acta. 1995 Sep 27;1252(1):15–22. doi: 10.1016/0167-4838(95)00052-v. [DOI] [PubMed] [Google Scholar]
  14. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Monzingo A. F., Robertus J. D. X-ray analysis of substrate analogs in the ricin A-chain active site. J Mol Biol. 1992 Oct 20;227(4):1136–1145. doi: 10.1016/0022-2836(92)90526-p. [DOI] [PubMed] [Google Scholar]
  16. Morris K. N., Wool I. G. Determination by systematic deletion of the amino acids essential for catalysis by ricin A chain. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):4869–4873. doi: 10.1073/pnas.89.11.4869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nicolas E., Beggs J. M., Haltiwanger B. M., Taraschi T. F. A new class of DNA glycosylase/apurinic/apyrimidinic lyases that act on specific adenines in single-stranded DNA. J Biol Chem. 1998 Jul 3;273(27):17216–17220. doi: 10.1074/jbc.273.27.17216. [DOI] [PubMed] [Google Scholar]
  18. Nicolas E., Beggs J. M., Haltiwanger B. M., Taraschi T. F. Direct evidence for the deoxyribonuclease activity of the plant ribosome inactivating protein gelonin. FEBS Lett. 1997 Apr 7;406(1-2):162–164. doi: 10.1016/s0014-5793(97)00267-6. [DOI] [PubMed] [Google Scholar]
  19. Peattie D. A. Direct chemical method for sequencing RNA. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1760–1764. doi: 10.1073/pnas.76.4.1760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Peumans W. J., Hao Q., Van Damme E. J. Ribosome-inactivating proteins from plants: more than RNA N-glycosidases? FASEB J. 2001 Jul;15(9):1493–1506. doi: 10.1096/fj.00-0751rev. [DOI] [PubMed] [Google Scholar]
  21. Pu Z., Xie L., Wang E., Liu W. Y. Purification and activity study of the A- and B-chains of cinnamomin, a type II ribosome-inactivating protein. Biol Chem. 1998 Dec;379(12):1413–1418. doi: 10.1515/bchm.1998.379.12.1413. [DOI] [PubMed] [Google Scholar]
  22. Roncuzzi L., Gasperi-Campani A. DNA-nuclease activity of the single-chain ribosome-inactivating proteins dianthin 30, saporin 6 and gelonin. FEBS Lett. 1996 Aug 19;392(1):16–20. doi: 10.1016/0014-5793(96)00776-4. [DOI] [PubMed] [Google Scholar]
  23. Tahirov T. H., Lu T. H., Liaw Y. C., Chen Y. L., Lin J. Y. Crystal structure of abrin-a at 2.14 A. J Mol Biol. 1995 Jul 14;250(3):354–367. doi: 10.1006/jmbi.1995.0382. [DOI] [PubMed] [Google Scholar]
  24. Wang Bao-Zhong, Liu Wang-Yi. Refolding of partially thermo-unfolded cinnamomin A-chain mediated by B-chain. Biochem Biophys Res Commun. 2003 Jun 20;306(1):39–45. doi: 10.1016/s0006-291x(03)00915-x. [DOI] [PubMed] [Google Scholar]
  25. Wang P., Tumer N. E. Pokeweed antiviral protein cleaves double-stranded supercoiled DNA using the same active site required to depurinate rRNA. Nucleic Acids Res. 1999 Apr 15;27(8):1900–1905. doi: 10.1093/nar/27.8.1900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Xie L., Wang B. Z., Hu R. G., Ji H. B., Zhang L., Liu W. Y. Structural and functional studies of cinnamomin, a new type II ribosome-inactivating protein isolated from the seeds of the camphor tree. Eur J Biochem. 2001 Nov;268(22):5723–5733. doi: 10.1046/j.0014-2956.2001.02515.x. [DOI] [PubMed] [Google Scholar]
  27. Yang Qiang, Liu Ren-shui, Gong Zhen-zhen, Liu Wang-Yi. Studies of three genes encoding Cinnamomin (a type II RIP) isolated from the seeds of camphor tree and their expression patterns. Gene. 2002 Feb 6;284(1-2):215–223. doi: 10.1016/s0378-1119(01)00890-3. [DOI] [PubMed] [Google Scholar]
  28. Zamboni M., Brigotti M., Rambelli F., Montanaro L., Sperti S. High-pressure-liquid-chromatographic and fluorimetric methods for the determination of adenine released from ribosomes by ricin and gelonin. Biochem J. 1989 May 1;259(3):639–643. doi: 10.1042/bj2590639. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES