RBF정제단백질의 핵산결합도 및 PKR효소의 인산화억제효과의 비교에 관한 연구

Comparative Study of Nucletic Acid Binding of the Purified RBF Protein and Its Inhibition of PKR phosphorylation

  • 박희성 (대구효성카톨릭대학교 식물육종학과) ;
  • 김인수 (경북대학교 유전공학과)
  • 발행 : 1998.04.01

초록

dsRNA결합인자인 RBF단백질을 정제하여 이의 단일 또는 이중선의 RNA 또는 DNA 와의 결합도를 측정하였다ㅓ. RBF단백질은 이들과 각각 반응시켜 그 결합도는 SDS-PAGE에 의하여 비교관찰하였다. RBF단백질은 dsRNA와은 강한 결합력을 나타낸 반면 기타의 핵산구조에 대해서는 이러한 결과를 나타내지 못하였다. 인산화 실험의 결과, RBF단백질은 poly(I) : poly(C)의 존재하에서 사람 도는 쥐 모두로 부터의 PKR 효소의 자가인산화를 유사한 방식으로 억제하였다. 이는 다른 종류의 진핵세포생물에서 단백질합성조절을 위한 PKR과 RBF가 유사한 경쟁적 관련성을 유지하면서 존재함을 시사하고 있다.

Column-purified double-stranded RNA binding factor (RBF) protein was tested for its binding affinity for the different forms of nucleic acids structure such as single-stranded(ss) and double-stranded(ds)RNA and ss- and dsDNA. The RBF protein was incubated with each of these nucleic acid structures in separate reactions and its comparative binding affnity was visualized by SDS-polyacrylamide gel electrophoresis. The RBF protein bound to the dsRNA molecule to form a tight RNA:protein complex in agreement with previous studies, but not to the other nucleic acid molecules confirming its distinctive affinity for the dsRNA structure. In phosphorylation assay in vito, the purified RBF protein significantly inhibited the autophosphorylation of the PKR derived from not only human but mouse source in the presence of poly(I):poly(C). It is suggesting that PKR vs. RBF is similarly under a competitive interaction among different eukaryotic organisms during protein synthesis.

키워드

참고문헌

  1. Genes & Dev. v.3 RNA-binding proteins as developmental regulators Bandziulis, R. M.;Swanson, M. S.;Dreyfuss, G.
  2. Mol. Biol. Rep. v.14 A seletive revies of RNA-protein interactions in eukaryotes Mattaj, I. W.
  3. Cell v.59 Tat trans-activates the human immunodeficiency virus through a nascent RNA target Berkout, B.;Silverman, R. H.;Jeang, K. T.
  4. Cell v.59 Sequence-specific recognition of RNA hairpins by bacteriophage antitermination requires a cinserved arginine-rich motif Lazinski, D. E.;Grzadzielska, E.;Das, A.
  5. Mol. Cell. Biol. v.11 Moleculat analysis of two poly(A) site-processing factors that determine the recognition and efficiency of cleavage of the premRNA Gilmartin, G.;Nevins, J.
  6. Science v.240 Iron-responsive elements : regulatory RNA sequences that control mRNA levels and translation Casy, J. L.;Hentze, M. W.;Koeller, D. M.;Caughman, S. W.;Rouault, T. A.;Klausner, R. D.;Harford, J. B.
  7. Cell v.68 Phosphorylation of initiation factor 2 alpha by protein kinase GCN2 mediates gene-specific translational control of GCN4 in yeast Dever, T. E.;Feng, L.;Wek, R. C.;Cigan, A. M.;Donahue, T. F;Hinnenbusch, A. G.
  8. Nucleic Acids Res. v.19 Detailed mutational analysis of Tar RNA : critical spacing beteen the bulge and loop recignition domains Berkout, B.;Jeang, K. T.
  9. Genes & Dev. v.6 Two RNA binding motifs in the double-stranded RNA activated protein kinase, DAI Green, S. R.;Mathews, M. B.
  10. Annu. Rev. Biochem. v.60 Translational control in mammalian cells Hershey, J. W. B.
  11. Eur. J. Biochem. v.178 The binding of double-stranded RNA and adenovirus VAI RNA to the interferon-induced protein kinase Galabru, J.;Katze, M. G.;Robert, N.;Hovanessian, A. G.
  12. Virology v.172 Mechanism fo interferon action. Activation of the human P1/eIF-2 alpha protein kinase by indivudual reovirus to s4 mRNA Bishoff, J.;Samuel, C. E.
  13. EMBO J. v.11 Human p68 kinase exhibits growth suppression in yeast and homology to the teanslational regulator GCN2 Chong, K. L.;Feng, L.;Schappert, K.;Meurs, E.;Donahue, T. F.;Friesen, J. D.;Hovanessian, A. G.;Williams, R. B.
  14. Proc. Natl. Acad. Sci. USA v.91 TAR RNAbinding protein is an inhibitor of the interferon-induced protein kinase PKR Park, H.;Davies, M. V.;Langland, J. O.;Chang, H.-W.;Nam, Y. S.;Tartaglia, J.;Paoletti, E.;Jacobs, B. L.;Kaufman, R. J.;Venkatesan, S.
  15. E. Coil. Kor. J. Gen. v.19 Inhibition of PKR phosphorylation in vitro by LacZ-fused double-stranded RNA binding protein(RBF) produced from E. Park, H.;Choi, J.-W.
  16. Kor. J. Life Sci. v.6 RNA binding specificities of double-stranded RNA binding protein(RBF) as an inhinitor of PKR kinase Park, H.;Choi, J.-W.
  17. Molecular Cloning, A Labortory Manual(2nd ed.) Sambrook, J.;Frisch, E. F.;Maniatis, T.
  18. Proc. Natl. Acad. Sci. USA v.89 Identification of double stranded RNA binding domains in the interferon induced double stranded RNA activated p68 kinase Feng, G. S.;Chong, K.;Kumar, A.;Williams, B. R.
  19. Mol. Cell. Biol. v.11 Functional expression and RNA binding analysis of the interferon-induced, double-stranded RNA-activated, 68,000-Mr protein kinase in a cell free system Katze, M. G.;Wambach, M. M.;Wong, L.;Garfinkel, M.;Meurs, E.;Chong, K.;Williams, B. R.;Hovanessian, A. G.;Barber, G. N.
  20. J. Biol. Chem. v.267 Identification of the double-stranded RNA-binding domain of the human interferon-inducible protein kinase Patel, R. C.;Sen, G. C.
  21. Cell v.62 Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon Meurs, E.;Chong, K.;Galabru, J.;Thomas, N. S. B.;Kerr, I. M.;Williams, B. R. G.;Hobanessian, A. G.
  22. Proc. Natl. Acad. Sci. USA v.85 Detection of activated of double-stranded RNA-dependent protein of acticated of double-stranded RNA-dependent Protein kinase in 3T3-F442A Cells Petryshyn, R.;Chen, J. J.;London, I. M.