BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
권호 표지
DOI QR코드

DOI QR Code

AUA as a Translation Initiation Site In Vitro for the Human Transcription Factor Sp3

  • Hernandez, Eric Moore (Department of Microbiology and Immunology, Georgetown University Medical Center) ;
  • Johnson, Anna (Department of Microbiology and Immunology, Georgetown University Medical Center) ;
  • Notario, Vicente (Department of Radiation Medicine, Georgetown University Medical Center) ;
  • Chen, Andrew (Department of Microbiology and Immunology, Georgetown University Medical Center) ;
  • Richert, John R. (Department of Microbiology and Immunology, Georgetown University Medical Center)
  • Published : 2002.05.31
Download PDF
⟨ Previous Next ⟩

Abstract

Sp3 is a bifunctional transcription factor that has been reported to stimulate or repress the transcription of numerous genes. Although the size of Sp3 mRNA is 4.0kb, the size of the known Sp3 cDNA sequence is 3.6kb. Thus, Sp3 functional studies have been performed with an artificially introduced start codon, and thus an amino-terminus that differs from the wild-type. Ideally, full-length cDNA expression vectors with the appropriate start codon should be utilized for these studies. Using 5'rapid amplification of cDNA ends, a full-length Sp3 cDNA clone was generated and the sequence verified in nine cell lines. No AUG initiation codon was present. However, stop codons were present in all three frames 5' to the known coding sequence. In vitro translation of this full-length cDNA clone produced the expected three isoforms-one at 100 kDa and two in the mid 60 kDa range. Electrophoretic mobility shift assays showed that the protein products had the ability to bind to the Sp1/3 consensus sequence. In vitro studies, using our Sp3 clone and site directed mutagenesis, identified the translation initiation site for the larger isoform as AUA. AUA has not been previously described as an endogenous initiation codon in eukaryotes.

Keywords

References

  1. Du, X. L., Edelstein, D., Rossetti, L., Fantus, I. G., Goldberg, H., Ziyadeh, F., Wu, J. and Brownlee, M. (2000) Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation. Proc. Natl. Acad. Sci. USA 97, 12222-12226. https://doi.org/10.1073/pnas.97.22.12222
  2. Gao, J. and Tsebg, L. (1996) Distal Sp3 binding sites in the hIGBP-1 gene promoter suppress transcriptional repression in decidualized human endometrial stromal cells: identification of a novel Sp3 form in decidual cells. Mol. Endocrinol. 10, 613-621. https://doi.org/10.1210/me.10.6.613
  3. Grekova, M. C., Robinson, E. D., Faerber, M. A., Katz, P., McFarland, H. F. and Richert, J. R. (1996) Deficient expression in multiple sclerosis of the inhibitory transcription factor Sp3 in mononuclear blood cells. Ann. Neurol. 40, 108-112. https://doi.org/10.1002/ana.410400117
  4. Hagen, G., Muller, S., Beato, M. and Suske, G. (1992) Cloning by recognition site screening of two novel GT box binding proteins: a family of Sp1 related genes. Nucleic Acids Res. 20, 5519-5525. https://doi.org/10.1093/nar/20.21.5519
  5. Hann, S. R. (1994) Regulation and function of non-AUG-initiated proto-oncogenes. Biochimie 76, 880-886. https://doi.org/10.1016/0300-9084(94)90190-2
  6. Kellogg, D. A., Doctor, B. P., Loebel, J. E. and Nirenberg, M. W. (1966) Synonym codon recognition by multiple species of valine-, alanine-, and methionine-sRNA. Proc. Natl. Acad. Sci. USA 55, 912-919. https://doi.org/10.1073/pnas.55.4.912
  7. Kennett, S. B., Udvadia, A. J. and Horowitz, J. M. (1997) Sp3 encodes multiple proteins that differ in their capacity to stimulate or repress transcription. Nucleic Acids Res. 25, 3110- 3117. https://doi.org/10.1093/nar/25.15.3110
  8. Kingsley, C. and Winoto, A. (1992) Cloning of GT box binding proteins: a novel Sp1 multigene family regulating T-cell receptor gene expression. Mol. Cell. Biol. 12, 4251-4261. https://doi.org/10.1128/MCB.12.10.4251
  9. Kozak, M. (1987) An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148. https://doi.org/10.1093/nar/15.20.8125
  10. Kozak, M. (1990) Downstream secondary structure facilitates recognition of initiator codons by eukaryotic ribosomes. Proc. Natl. Acad. Sci. USA 87, 8301-8305. https://doi.org/10.1073/pnas.87.21.8301
  11. Majello, B., De Luca, P. and Lania, L. (1997) Sp3 is a bifunctional transcription regulator with modular independent activation and repression domains. J. Biol. Chem. 272, 4021-4026. https://doi.org/10.1074/jbc.272.7.4021
  12. Mehdi, H., Ono, E. and Gupta, K. C. (1990) Initiation of translation at CUG, GUG, and ACG codons in mammalian cells. Gene 91, 173-178. https://doi.org/10.1016/0378-1119(90)90085-6
  13. Melnikova, I. N., Yang, Y. and Gardner, P. D. (2000) Interactions between regulatory proteins that bind to the nicotinic receptor beta4 subunit gene promoter. Eur. J. Pharmacol. 393, 75-83. https://doi.org/10.1016/S0014-2999(99)00884-5
  14. Pan, X., Solomon, S. S., Shah, R. J., Palazzolo, M. R. and Raghow, R. S. (2000) Members of the Sp transcription factor family regulate rat calmodulin gene expression. J. Lab. Clin. Med. 136, 157-163. https://doi.org/10.1067/mlc.2000.108149
  15. Peabody, D. S. (1989) Translation initiation at non-AUG triplets in mammalian cells. J. Biol. Chem. 264, 5031-5035.
  16. Redell, J. B. and Tempel, B. L. (1998) Multiple promoter elements interact to control the transcription of the potassium channel gene, KCNJ2. J. Biol. Chem. 273, 22807-22818. https://doi.org/10.1074/jbc.273.35.22807
  17. Suske, G. (1999) The Sp-family of Transcription factors. Gene 238, 291-300. https://doi.org/10.1016/S0378-1119(99)00357-1
  18. Tone, M., Powell, M. J., Tone, Y., Thompson, S. A. and Waldmann, H. (2000) IL-10 gene expression is controlled by the transcription factors Sp1 and Sp3. J. Immunol. 165, 286-291. https://doi.org/10.4049/jimmunol.165.1.286
  19. Tymms, M. J. (1995) Methods in Molecular Biology; In Vitro Transcription and Translation Protocols, vol 37, Humana Press Inc, Totowa, New Jersy.
  20. Walter, P. and Blobel, G. (1983) Preparation of microsomal membranes for co-translational protein translocation. Methods Enzymol. 96, 84-93. https://doi.org/10.1016/S0076-6879(83)96010-X
  21. Yang, Y., Hwang, C. K., Jun, E., Lee, G. and Mouradian, M. M. (2000) ZIC2 and Sp3 repress Sp1-induced activation of the human D1A dopamine receptor gene. J. Biol. Chem. 49, 38863-38869.

Cited by

  1. 6-mercaptopurine influences TPMT gene transcription in a TPMT gene promoter variable number of tandem repeats-dependent manner vol.13, pp.3, 2012, https://doi.org/10.2217/pgs.11.153
  2. 5' Genomic Structure of Human Sp3 vol.19, pp.11, 2002, https://doi.org/10.1093/oxfordjournals.molbev.a004026
  3. Human transcription factor Sp3: genomic structure, identification of a processed pseudogene, and transcript analysis vol.341, 2004, https://doi.org/10.1016/j.gene.2004.06.055
  4. Mua-6, a gene required for tissue integrity in Caenorhabditis elegans, encodes a cytoplasmic intermediate filament vol.263, pp.2, 2003, https://doi.org/10.1016/j.ydbio.2003.08.001