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. 1991 Nov 25;19(22):6197–6203. doi: 10.1093/nar/19.22.6197

High yield purification of active transcription factor IIIA expressed in E. coli.

S Del Río 1, D R Setzer 1
PMCID: PMC329122  PMID: 1956778

Abstract

Transcription factor IIIA (TFIIIA), a sequence-specific DNA-binding protein from Xenopus laevis, is a zinc finger protein required for transcription of 5S rRNA genes by RNA polymerase III. We describe the purification and characterization of recombinant TFIIIA (recTFIIIA) expressed in E. coli. RecTFIIIA was purified to greater than 95% homogeneity at a yield of 2-3 milligrams per liter of bacterial culture. This purified protein protects the internal control region of a 5S rRNA gene from DNase I digestion, yielding footprints on both strands identical to those produced by the ovarian protein (ovaTFIIIA). Quantitative analysis of binding data from gel retardation assays yielded a KD of about 0.4 nM for TFIIIA from either source. Using a quantitative TFIIIA-dependent in vitro transcription assay, we found that recTFIIIA is equivalent to ovaTFIIIA in supporting transcription of 5S rRNA genes. We conclude that recTFIIIA is functionally indistinguishable from the protein purified from Xenopus ovaries, and can be readily obtained in pure form and large quantity.

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  1. Bieker J. J., Roeder R. G. Physical properties and DNA-binding stoichiometry of a 5 S gene-specific transcription factor. J Biol Chem. 1984 May 25;259(10):6158–6164. [PubMed] [Google Scholar]
  2. Bogenhagen D. F., Sakonju S., Brown D. D. A control region in the center of the 5S RNA gene directs specific initiation of transcription: II. The 3' border of the region. Cell. 1980 Jan;19(1):27–35. doi: 10.1016/0092-8674(80)90385-2. [DOI] [PubMed] [Google Scholar]
  3. Bogenhagen D. F., Wormington W. M., Brown D. D. Stable transcription complexes of Xenopus 5S RNA genes: a means to maintain the differentiated state. Cell. 1982 Feb;28(2):413–421. doi: 10.1016/0092-8674(82)90359-2. [DOI] [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. Campbell F. E., Jr, Setzer D. R. Displacement of Xenopus transcription factor IIIA from a 5S rRNA gene by a transcribing RNA polymerase. Mol Cell Biol. 1991 Aug;11(8):3978–3986. doi: 10.1128/mcb.11.8.3978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dignam J. D., Martin P. L., Shastry B. S., Roeder R. G. Eukaryotic gene transcription with purified components. Methods Enzymol. 1983;101:582–598. doi: 10.1016/0076-6879(83)01039-3. [DOI] [PubMed] [Google Scholar]
  7. Engelke D. R., Ng S. Y., Shastry B. S., Roeder R. G. Specific interaction of a purified transcription factor with an internal control region of 5S RNA genes. Cell. 1980 Mar;19(3):717–728. doi: 10.1016/s0092-8674(80)80048-1. [DOI] [PubMed] [Google Scholar]
  8. Fairall L., Rhodes D., Klug A. Mapping of the sites of protection on a 5 S RNA gene by the Xenopus transcription factor IIIA. A model for the interaction. J Mol Biol. 1986 Dec 5;192(3):577–591. doi: 10.1016/0022-2836(86)90278-0. [DOI] [PubMed] [Google Scholar]
  9. Fiser-Littell R. M., Duke A. L., Yanchick J. S., Hanas J. S. Deletion of the N-terminal region of Xenopus transcription factor IIIA inhibits specific binding to the 5 S RNA gene. J Biol Chem. 1988 Feb 5;263(4):1607–1610. [PubMed] [Google Scholar]
  10. Fradkin L. G., Yoshinaga S. K., Berk A. J., Dasgupta A. Human transcription factor TFIIIC2 specifically interacts with a unique sequence in the Xenopus laevis 5S rRNA gene. Mol Cell Biol. 1989 Nov;9(11):4941–4950. doi: 10.1128/mcb.9.11.4941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ginsberg A. M., King B. O., Roeder R. G. Xenopus 5S gene transcription factor, TFIIIA: characterization of a cDNA clone and measurement of RNA levels throughout development. Cell. 1984 Dec;39(3 Pt 2):479–489. doi: 10.1016/0092-8674(84)90455-0. [DOI] [PubMed] [Google Scholar]
  12. Gough J. A., Murray N. E. Sequence diversity among related genes for recognition of specific targets in DNA molecules. J Mol Biol. 1983 May 5;166(1):1–19. doi: 10.1016/s0022-2836(83)80047-3. [DOI] [PubMed] [Google Scholar]
  13. Gouy M., Gautier C. Codon usage in bacteria: correlation with gene expressivity. Nucleic Acids Res. 1982 Nov 25;10(22):7055–7074. doi: 10.1093/nar/10.22.7055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hanas J. S., Hazuda D. J., Bogenhagen D. F., Wu F. Y., Wu C. W. Xenopus transcription factor A requires zinc for binding to the 5 S RNA gene. J Biol Chem. 1983 Dec 10;258(23):14120–14125. [PubMed] [Google Scholar]
  15. Hanas J. S., Littell R. M., Gaskins C. J., Zebrowski R. Internal deletion mutants of Xenopus transcription factor IIIA. Nucleic Acids Res. 1989 Dec 11;17(23):9861–9870. doi: 10.1093/nar/17.23.9861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hayes J., Tullius T. D., Wolffe A. P. A protein-protein interaction is essential for stable complex formation on a 5 S RNA gene. J Biol Chem. 1989 Apr 15;264(11):6009–6012. [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Lassar A. B., Martin P. L., Roeder R. G. Transcription of class III genes: formation of preinitiation complexes. Science. 1983 Nov 18;222(4625):740–748. doi: 10.1126/science.6356356. [DOI] [PubMed] [Google Scholar]
  19. Miller J., McLachlan A. D., Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985 Jun;4(6):1609–1614. doi: 10.1002/j.1460-2075.1985.tb03825.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ottonello S., Rivier D. H., Doolittle G. M., Young L. S., Sprague K. U. The properties of a new polymerase III transcription factor reveal that transcription complexes can assemble by more than one pathway. EMBO J. 1987 Jul;6(7):1921–1927. doi: 10.1002/j.1460-2075.1987.tb02452.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pao C. I., Lee T. C., Liao Y. D., Wu C. W. An N-terminally fused Xenopus transcription factor IIIA synthesized in Escherichia coli is biologically active. J Biol Chem. 1988 Jul 25;263(21):10295–10299. [PubMed] [Google Scholar]
  22. Pelham H. R., Brown D. D. A specific transcription factor that can bind either the 5S RNA gene or 5S RNA. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4170–4174. doi: 10.1073/pnas.77.7.4170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Picard B., Wegnez M. Isolation of a 7S particle from Xenopus laevis oocytes: a 5S RNA-protein complex. Proc Natl Acad Sci U S A. 1979 Jan;76(1):241–245. doi: 10.1073/pnas.76.1.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Robinson M., Lilley R., Little S., Emtage J. S., Yarranton G., Stephens P., Millican A., Eaton M., Humphreys G. Codon usage can affect efficiency of translation of genes in Escherichia coli. Nucleic Acids Res. 1984 Sep 11;12(17):6663–6671. doi: 10.1093/nar/12.17.6663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sakonju S., Brown D. D., Engelke D., Ng S. Y., Shastry B. S., Roeder R. G. The binding of a transcription factor to deletion mutants of a 5S ribosomal RNA gene. Cell. 1981 Mar;23(3):665–669. doi: 10.1016/0092-8674(81)90429-3. [DOI] [PubMed] [Google Scholar]
  26. Segall J., Matsui T., Roeder R. G. Multiple factors are required for the accurate transcription of purified genes by RNA polymerase III. J Biol Chem. 1980 Dec 25;255(24):11986–11991. [PubMed] [Google Scholar]
  27. Setzer D. R., Brown D. D. Formation and stability of the 5 S RNA transcription complex. J Biol Chem. 1985 Feb 25;260(4):2483–2492. [PubMed] [Google Scholar]
  28. Setzer D. R., Hmiel R. M., Liao S. Y. A simple vector modification to facilitate oligonucleotide-directed mutagenesis. Nucleic Acids Res. 1990 Jul 25;18(14):4175–4178. doi: 10.1093/nar/18.14.4175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Shastry B. S., Ng S. Y., Roeder R. G. Multiple factors involved in the transcription of class III genes in Xenopus laevis. J Biol Chem. 1982 Nov 10;257(21):12979–12986. [PubMed] [Google Scholar]
  30. Smith D. R., Jackson I. J., Brown D. D. Domains of the positive transcription factor specific for the Xenopus 5S RNA gene. Cell. 1984 Jun;37(2):645–652. doi: 10.1016/0092-8674(84)90396-9. [DOI] [PubMed] [Google Scholar]
  31. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  32. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  33. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Effect of pyrophosphorolysis and metal ions. J Biol Chem. 1990 May 15;265(14):8322–8328. [PubMed] [Google Scholar]
  34. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tso J. Y., Siebel C., Korn L. J. Expression of functional Xenopus TFIIIA in Escherichia coli. Nucleic Acids Res. 1989 Mar 11;17(5):2145–2145. doi: 10.1093/nar/17.5.2145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wolffe A. P. Transcription fraction TFIIIC can regulate differential Xenopus 5S RNA gene transcription in vitro. EMBO J. 1988 Apr;7(4):1071–1079. doi: 10.1002/j.1460-2075.1988.tb02915.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]
  38. Young L. S., Dunstan H. M., Witte P. R., Smith T. P., Ottonello S., Sprague K. U. A class III transcription factor composed of RNA. Science. 1991 Apr 26;252(5005):542–546. doi: 10.1126/science.1708526. [DOI] [PubMed] [Google Scholar]
  39. Zwieb C., Brown R. S. Absence of substantial bending in Xenopus laevis transcription factor IIIA-DNA complexes. Nucleic Acids Res. 1990 Feb 11;18(3):583–587. doi: 10.1093/nar/18.3.583. [DOI] [PMC free article] [PubMed] [Google Scholar]

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