Skip to main content
PMC home page
RNA logoLink to RNA
. 1998 Feb;4(2):205–214.

ATP is a cofactor of the Upf1 protein that modulates its translation termination and RNA binding activities.

Y Weng 1, K Czaplinski 1, S W Peltz 1
PMCID: PMC1369609  PMID: 9570320

Abstract

The nonsense-mediated mRNA decay pathway decreases the abundance of mRNAs that contain premature termination codons and prevents suppression of nonsense alleles. The UPF1 gene in the yeast Saccharomyces cerevisiae was shown to be a trans-acting factor in this decay pathway. The Upf1p demonstrates RNA-dependent ATPase, RNA helicase, and RNA binding activities. The results presented here investigate the binding affinity of the Upf1p for ATP and the consequences of ATP binding on its affinity for RNA. The results demonstrate that the Upf1p binds ATP in the absence of RNA. Consistent with this result, the TR800AA mutant form of the Upf1p still bound ATP, although it does not bind RNA. ATP binding also modulates the affinity of Upf1p for RNA. The RNA binding activity of the DE572AA mutant form of the Upf1p, which lacks ATPase activity, still bound ATP as efficiently as the wild-type Upf1p and destabilized the Upf1p-RNA complex. Similarly, ATPgammaS, a nonhydrolyzable analogue of ATP, interacted with Upf1p and promoted disassociation of the Upf1p-RNA complex. The conserved lysine residue (K436) in the helicase motif Ia in the Upf1p was shown to be critical for ATP binding. Taken together, these findings formally prove that ATP can bind Upf1p in the absence of RNA and that this interaction has consequences on the formation of the Upf1p-RNA complex. Further, the results support the genetic evidence indicating that ATP binding is important for the Upf1p to increase the translation termination efficiency at a nonsense codon. Based on these findings, a model describing how the Upf1p functions in modulating translation and turnover and the potential insights into the mechanism of the Upf1p helicase will be discussed.

Full Text

The Full Text of this article is available as a PDF (497.1 KB).

Selected References

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

  1. Atkin A. L., Altamura N., Leeds P., Culbertson M. R. The majority of yeast UPF1 co-localizes with polyribosomes in the cytoplasm. Mol Biol Cell. 1995 May;6(5):611–625. doi: 10.1091/mbc.6.5.611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brosh R. M., Jr, Matson S. W. Mutations in motif II of Escherichia coli DNA helicase II render the enzyme nonfunctional in both mismatch repair and excision repair with differential effects on the unwinding reaction. J Bacteriol. 1995 Oct;177(19):5612–5621. doi: 10.1128/jb.177.19.5612-5621.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chao K. L., Lohman T. M. DNA-induced dimerization of the Escherichia coli Rep helicase. J Mol Biol. 1991 Oct 20;221(4):1165–1181. doi: 10.1016/0022-2836(91)90926-w. [DOI] [PubMed] [Google Scholar]
  4. Cui Y., Dinman J. D., Peltz S. W. Mof4-1 is an allele of the UPF1/IFS2 gene which affects both mRNA turnover and -1 ribosomal frameshifting efficiency. EMBO J. 1996 Oct 15;15(20):5726–5736. doi: 10.1002/j.1460-2075.1996.tb00956.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cui Y., Hagan K. W., Zhang S., Peltz S. W. Identification and characterization of genes that are required for the accelerated degradation of mRNAs containing a premature translational termination codon. Genes Dev. 1995 Feb 15;9(4):423–436. doi: 10.1101/gad.9.4.423. [DOI] [PubMed] [Google Scholar]
  6. Czaplinski K., Weng Y., Hagan K. W., Peltz S. W. Purification and characterization of the Upf1 protein: a factor involved in translation and mRNA degradation. RNA. 1995 Aug;1(6):610–623. [PMC free article] [PubMed] [Google Scholar]
  7. Decker C. J., Parker R. Mechanisms of mRNA degradation in eukaryotes. Trends Biochem Sci. 1994 Aug;19(8):336–340. doi: 10.1016/0968-0004(94)90073-6. [DOI] [PubMed] [Google Scholar]
  8. Fry D. C., Kuby S. A., Mildvan A. S. ATP-binding site of adenylate kinase: mechanistic implications of its homology with ras-encoded p21, F1-ATPase, and other nucleotide-binding proteins. Proc Natl Acad Sci U S A. 1986 Feb;83(4):907–911. doi: 10.1073/pnas.83.4.907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res. 1989 Jun 26;17(12):4713–4730. doi: 10.1093/nar/17.12.4713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. He F., Brown A. H., Jacobson A. Interaction between Nmd2p and Upf1p is required for activity but not for dominant-negative inhibition of the nonsense-mediated mRNA decay pathway in yeast. RNA. 1996 Feb;2(2):153–170. [PMC free article] [PubMed] [Google Scholar]
  11. He F., Brown A. H., Jacobson A. Upf1p, Nmd2p, and Upf3p are interacting components of the yeast nonsense-mediated mRNA decay pathway. Mol Cell Biol. 1997 Mar;17(3):1580–1594. doi: 10.1128/mcb.17.3.1580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. He F., Jacobson A. Identification of a novel component of the nonsense-mediated mRNA decay pathway by use of an interacting protein screen. Genes Dev. 1995 Feb 15;9(4):437–454. doi: 10.1101/gad.9.4.437. [DOI] [PubMed] [Google Scholar]
  13. He F., Peltz S. W., Donahue J. L., Rosbash M., Jacobson A. Stabilization and ribosome association of unspliced pre-mRNAs in a yeast upf1- mutant. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7034–7038. doi: 10.1073/pnas.90.15.7034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jacobson A., Peltz S. W. Interrelationships of the pathways of mRNA decay and translation in eukaryotic cells. Annu Rev Biochem. 1996;65:693–739. doi: 10.1146/annurev.bi.65.070196.003401. [DOI] [PubMed] [Google Scholar]
  15. Jurnak F. The three-dimensional structure of c-H-ras p21: implications for oncogene and G protein studies. Trends Biochem Sci. 1988 Jun;13(6):195–198. doi: 10.1016/0968-0004(88)90080-1. [DOI] [PubMed] [Google Scholar]
  16. Koonin E. V. A new group of putative RNA helicases. Trends Biochem Sci. 1992 Dec;17(12):495–497. doi: 10.1016/0968-0004(92)90338-a. [DOI] [PubMed] [Google Scholar]
  17. Lee B. S., Culbertson M. R. Identification of an additional gene required for eukaryotic nonsense mRNA turnover. Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10354–10358. doi: 10.1073/pnas.92.22.10354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lee C. G., Hurwitz J. A new RNA helicase isolated from HeLa cells that catalytically translocates in the 3' to 5' direction. J Biol Chem. 1992 Mar 5;267(7):4398–4407. [PubMed] [Google Scholar]
  19. Leeds P., Peltz S. W., Jacobson A., Culbertson M. R. The product of the yeast UPF1 gene is required for rapid turnover of mRNAs containing a premature translational termination codon. Genes Dev. 1991 Dec;5(12A):2303–2314. doi: 10.1101/gad.5.12a.2303. [DOI] [PubMed] [Google Scholar]
  20. Leeds P., Wood J. M., Lee B. S., Culbertson M. R. Gene products that promote mRNA turnover in Saccharomyces cerevisiae. Mol Cell Biol. 1992 May;12(5):2165–2177. doi: 10.1128/mcb.12.5.2165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Linder P., Lasko P. F., Ashburner M., Leroy P., Nielsen P. J., Nishi K., Schnier J., Slonimski P. P. Birth of the D-E-A-D box. Nature. 1989 Jan 12;337(6203):121–122. doi: 10.1038/337121a0. [DOI] [PubMed] [Google Scholar]
  22. Lohman T. M., Bjornson K. P. Mechanisms of helicase-catalyzed DNA unwinding. Annu Rev Biochem. 1996;65:169–214. doi: 10.1146/annurev.bi.65.070196.001125. [DOI] [PubMed] [Google Scholar]
  23. Maquat L. E. When cells stop making sense: effects of nonsense codons on RNA metabolism in vertebrate cells. RNA. 1995 Jul;1(5):453–465. [PMC free article] [PubMed] [Google Scholar]
  24. Pause A., Méthot N., Sonenberg N. The HRIGRXXR region of the DEAD box RNA helicase eukaryotic translation initiation factor 4A is required for RNA binding and ATP hydrolysis. Mol Cell Biol. 1993 Nov;13(11):6789–6798. doi: 10.1128/mcb.13.11.6789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pause A., Sonenberg N. Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A. EMBO J. 1992 Jul;11(7):2643–2654. doi: 10.1002/j.1460-2075.1992.tb05330.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ross J. mRNA stability in mammalian cells. Microbiol Rev. 1995 Sep;59(3):423–450. doi: 10.1128/mr.59.3.423-450.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ruiz-Echevarria M. J., Czaplinski K., Peltz S. W. Making sense of nonsense in yeast. Trends Biochem Sci. 1996 Nov;21(11):433–438. doi: 10.1016/s0968-0004(96)10055-4. [DOI] [PubMed] [Google Scholar]
  28. Stansfield I., Jones K. M., Kushnirov V. V., Dagkesamanskaya A. R., Poznyakovski A. I., Paushkin S. V., Nierras C. R., Cox B. S., Ter-Avanesyan M. D., Tuite M. F. The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae. EMBO J. 1995 Sep 1;14(17):4365–4373. doi: 10.1002/j.1460-2075.1995.tb00111.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Weng Y., Czaplinski K., Peltz S. W. Genetic and biochemical characterization of mutations in the ATPase and helicase regions of the Upf1 protein. Mol Cell Biol. 1996 Oct;16(10):5477–5490. doi: 10.1128/mcb.16.10.5477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Weng Y., Czaplinski K., Peltz S. W. Identification and characterization of mutations in the UPF1 gene that affect nonsense suppression and the formation of the Upf protein complex but not mRNA turnover. Mol Cell Biol. 1996 Oct;16(10):5491–5506. doi: 10.1128/mcb.16.10.5491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wong I., Lohman T. M. Allosteric effects of nucleotide cofactors on Escherichia coli Rep helicase-DNA binding. Science. 1992 Apr 17;256(5055):350–355. doi: 10.1126/science.256.5055.350. [DOI] [PubMed] [Google Scholar]
  33. Yun D. F., Sherman F. Initiation of translation can occur only in a restricted region of the CYC1 mRNA of Saccharomyces cerevisiae. Mol Cell Biol. 1995 Feb;15(2):1021–1033. doi: 10.1128/mcb.15.2.1021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. de Vos A. M., Tong L., Milburn M. V., Matias P. M., Jancarik J., Noguchi S., Nishimura S., Miura K., Ohtsuka E., Kim S. H. Three-dimensional structure of an oncogene protein: catalytic domain of human c-H-ras p21. Science. 1988 Feb 19;239(4842):888–893. doi: 10.1126/science.2448879. [DOI] [PubMed] [Google Scholar]
  35. la Cour T. F., Nyborg J., Thirup S., Clark B. F. Structural details of the binding of guanosine diphosphate to elongation factor Tu from E. coli as studied by X-ray crystallography. EMBO J. 1985 Sep;4(9):2385–2388. doi: 10.1002/j.1460-2075.1985.tb03943.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES