Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1986 Dec;6(12):4723–4733. doi: 10.1128/mcb.6.12.4723

A single polypeptide possesses the binding and transcription activities of the adenovirus major late transcription factor.

L A Chodosh, R W Carthew, P A Sharp
PMCID: PMC367258  PMID: 3796614

Abstract

A simple approach has been developed for the unambiguous identification and purification of sequence-specific DNA-binding proteins solely on the basis of their ability to bind selectively to their target sequences. Four independent methods were used to identify the promoter-specific RNA polymerase II transcription factor MLTF as a 46-kilodalton (kDa) polypeptide. First, a 46-kDa protein was specifically cross-linked by UV irradiation to a body-labeled DNA fragment containing the MLTF binding site. Second, MLTF sedimented through glycerol gradients at a rate corresponding to a protein of native molecular weight 45,000 to 50,000. Third, a 46-kDa protein was specifically retained on a biotin-streptavidin matrix only when the DNA fragment coupled to the matrix contained the MLTF binding site. Finally, proteins from the most highly purified fraction which were eluted and renatured from the 44- to 48-kDa region of a sodium dodecyl sulfate-polyacrylamide gel exhibited both binding and transcription-stimulatory activities. The DNA-binding activity was purified 100,000-fold by chromatography through three conventional columns plus a DNA affinity column. Purified MLTF was characterized with respect to the kinetic and thermodynamic properties of DNA binding. These parameters indicate a high degree of occupancy of MLTF binding sites in vivo.

Full text

PDF
4723

Images in this article

Selected References

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

  1. 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]
  2. Brent R., Ptashne M. A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor. Cell. 1985 Dec;43(3 Pt 2):729–736. doi: 10.1016/0092-8674(85)90246-6. [DOI] [PubMed] [Google Scholar]
  3. Carthew R. W., Chodosh L. A., Sharp P. A. An RNA polymerase II transcription factor binds to an upstream element in the adenovirus major late promoter. Cell. 1985 Dec;43(2 Pt 1):439–448. doi: 10.1016/0092-8674(85)90174-6. [DOI] [PubMed] [Google Scholar]
  4. Dynan W. S., Tjian R. Isolation of transcription factors that discriminate between different promoters recognized by RNA polymerase II. Cell. 1983 Mar;32(3):669–680. doi: 10.1016/0092-8674(83)90053-3. [DOI] [PubMed] [Google Scholar]
  5. Dynan W. S., Tjian R. The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter. Cell. 1983 Nov;35(1):79–87. doi: 10.1016/0092-8674(83)90210-6. [DOI] [PubMed] [Google Scholar]
  6. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Grabowski P. J., Sharp P. A. Affinity chromatography of splicing complexes: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles in the spliceosome. Science. 1986 Sep 19;233(4770):1294–1299. doi: 10.1126/science.3638792. [DOI] [PubMed] [Google Scholar]
  8. Haeuptle M. T., Aubert M. L., Djiane J., Kraehenbuhl J. P. Binding sites for lactogenic and somatogenic hormones from rabbit mammary gland and liver. J Biol Chem. 1983 Jan 10;258(1):305–314. [PubMed] [Google Scholar]
  9. Hager D. A., Burgess R. R. Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem. 1980 Nov 15;109(1):76–86. doi: 10.1016/0003-2697(80)90013-5. [DOI] [PubMed] [Google Scholar]
  10. Hen R., Sassone-Corsi P., Corden J., Gaub M. P., Chambon P. Sequences upstream from the T-A-T-A box are required in vivo and in vitro for efficient transcription from the adenovirus serotype 2 major late promoter. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7132–7136. doi: 10.1073/pnas.79.23.7132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hillel Z., Wu C. W. Photochemical cross-linking studies on the interaction of Escherichia coli RNA polymerase with T7 DNA. Biochemistry. 1978 Jul 25;17(15):2954–2961. doi: 10.1021/bi00608a003. [DOI] [PubMed] [Google Scholar]
  12. Hochschild A., Irwin N., Ptashne M. Repressor structure and the mechanism of positive control. Cell. 1983 Feb;32(2):319–325. doi: 10.1016/0092-8674(83)90451-8. [DOI] [PubMed] [Google Scholar]
  13. Jove R., Manley J. L. In vitro transcription from the adenovirus 2 major late promoter utilizing templates truncated at promoter-proximal sites. J Biol Chem. 1984 Jul 10;259(13):8513–8521. [PubMed] [Google Scholar]
  14. 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]
  15. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  16. Lin S. Y., Riggs A. D. Photochemical attachment of lac repressor to bromodeoxyuridine-substituted lac operator by ultraviolet radiation. Proc Natl Acad Sci U S A. 1974 Mar;71(3):947–951. doi: 10.1073/pnas.71.3.947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lin S., Riggs A. D. The general affinity of lac repressor for E. coli DNA: implications for gene regulation in procaryotes and eucaryotes. Cell. 1975 Feb;4(2):107–111. doi: 10.1016/0092-8674(75)90116-6. [DOI] [PubMed] [Google Scholar]
  18. McKnight S. L., Kingsbury R. C., Spence A., Smith M. The distal transcription signals of the herpesvirus tk gene share a common hexanucleotide control sequence. Cell. 1984 May;37(1):253–262. doi: 10.1016/0092-8674(84)90321-0. [DOI] [PubMed] [Google Scholar]
  19. Miyamoto N. G., Moncollin V., Egly J. M., Chambon P. Specific interaction between a transcription factor and the upstream element of the adenovirus-2 major late promoter. EMBO J. 1985 Dec 16;4(13A):3563–3570. doi: 10.1002/j.1460-2075.1985.tb04118.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  21. Ogata R. T., Gilbert W. DNA-binding site of lac repressor probed by dimethylsulfate methylation of lac operator. J Mol Biol. 1979 Aug 25;132(4):709–728. doi: 10.1016/0022-2836(79)90384-x. [DOI] [PubMed] [Google Scholar]
  22. Parker C. S., Topol J. A Drosophila RNA polymerase II transcription factor contains a promoter-region-specific DNA-binding activity. Cell. 1984 Feb;36(2):357–369. doi: 10.1016/0092-8674(84)90229-0. [DOI] [PubMed] [Google Scholar]
  23. Piette J., Kryszke M. H., Yaniv M. Specific interaction of cellular factors with the B enhancer of polyoma virus. EMBO J. 1985 Oct;4(10):2675–2685. doi: 10.1002/j.1460-2075.1985.tb03987.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ptashne M., Jeffrey A., Johnson A. D., Maurer R., Meyer B. J., Pabo C. O., Roberts T. M., Sauer R. T. How the lambda repressor and cro work. Cell. 1980 Jan;19(1):1–11. doi: 10.1016/0092-8674(80)90383-9. [DOI] [PubMed] [Google Scholar]
  25. Record M. T., Jr, Lohman M. L., De Haseth P. Ion effects on ligand-nucleic acid interactions. J Mol Biol. 1976 Oct 25;107(2):145–158. doi: 10.1016/s0022-2836(76)80023-x. [DOI] [PubMed] [Google Scholar]
  26. Riggs A. D., Bourgeois S., Cohn M. The lac repressor-operator interaction. 3. Kinetic studies. J Mol Biol. 1970 Nov 14;53(3):401–417. doi: 10.1016/0022-2836(70)90074-4. [DOI] [PubMed] [Google Scholar]
  27. Riggs A. D., Bourgeois S. On the assay, isolation and characterization of the lac repressor. J Mol Biol. 1968 Jul 14;34(2):361–364. doi: 10.1016/0022-2836(68)90260-x. [DOI] [PubMed] [Google Scholar]
  28. Riggs A. D., Suzuki H., Bourgeois S. Lac repressor-operator interaction. I. Equilibrium studies. J Mol Biol. 1970 Feb 28;48(1):67–83. doi: 10.1016/0022-2836(70)90219-6. [DOI] [PubMed] [Google Scholar]
  29. Samuels M., Fire A., Sharp P. A. Dinucleotide priming of transcription mediated by RNA polymerase II. J Biol Chem. 1984 Feb 25;259(4):2517–2525. [PubMed] [Google Scholar]
  30. Samuels M., Fire A., Sharp P. A. Separation and characterization of factors mediating accurate transcription by RNA polymerase II. J Biol Chem. 1982 Dec 10;257(23):14419–14427. [PubMed] [Google Scholar]
  31. Samuels M., Sharp P. A. Purification and characterization of a specific RNA polymerase II transcription factor. J Biol Chem. 1986 Feb 15;261(5):2003–2013. [PubMed] [Google Scholar]
  32. Sawadogo M., Roeder R. G. Interaction of a gene-specific transcription factor with the adenovirus major late promoter upstream of the TATA box region. Cell. 1985 Nov;43(1):165–175. doi: 10.1016/0092-8674(85)90021-2. [DOI] [PubMed] [Google Scholar]
  33. Weyer U., Doerfler W. Species dependence of the major late promoter in adenovirus type 12 DNA. EMBO J. 1985 Nov;4(11):3015–3019. doi: 10.1002/j.1460-2075.1985.tb04037.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yu Y. T., Manley J. L. Generation and functional analyses for base-substitution mutants of the adenovirus 2 major late promoter. Nucleic Acids Res. 1984 Dec 21;12(24):9309–9321. doi: 10.1093/nar/12.24.9309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. von Hippel P. H., Bear D. G., Morgan W. D., McSwiggen J. A. Protein-nucleic acid interactions in transcription: a molecular analysis. Annu Rev Biochem. 1984;53:389–446. doi: 10.1146/annurev.bi.53.070184.002133. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES