Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1991 Oct;65(10):5297–5304. doi: 10.1128/jvi.65.10.5297-5304.1991

A purified adenovirus 289-amino-acid E1A protein activates RNA polymerase III transcription in vitro and alters transcription factor TFIIIC.

S Datta 1, C J Soong 1, D M Wang 1, M L Harter 1
PMCID: PMC249009  PMID: 1832723

Abstract

We have previously demonstrated that a purified bacterially synthesized E1A 289-amino-acid protein is capable of stimulating transcription from the promoters of genes transcribed by RNA polymerase II in vitro (R. Spangler, M. Bruner, B. Dalie, and M. L. Harter, Science 237:1044-1046, 1987). In this study, we show that this protein is also capable of transactivating in vitro the adenovirus virus-associated (VA1) RNA gene transcribed by RNA polymerase III. Pertinent to the transcription of this gene is the rate-limiting component, TFIIIC, which appears to be of two distinct forms in uninfected HeLa cells. The addition of an oligonucleotide containing a TFIIIC binding site to HeLa whole-cell extracts inhibits VA1 transcription by sequestering TFIIIC. However, the addition of purified E1A to extracts previously challenged with the TFIIIC oligonucleotide restores the level of VA1 transcription. When included in the same reaction, an E1A-specific monoclonal antibody reverses the restoration. Incubation of purified E1A with either HeLa cell nuclear or whole-cell extracts alters the DNA-binding properties of TFIIIC as detected by gel shift assays. This alteration does not occur if E1A-specific antibody and E1A protein are added simultaneously to the extract. In contrast, the addition of this antibody to extracts at a later time does not reverse the alteration observed in the TFIIIC binding activities. Never at any time did we note the formation of novel TFIIIC-promoter complexes after the addition of E1A to nuclear extracts. These results clearly establish that E1A mediates its effect on VA1 transcription through TFIIIC in a very rapid yet indirect manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Full text

PDF
5297

Images in this article

5299Image
on p.5299
5300Image
on p.5300
5300Image
on p.5300
5301Image
on p.5301
5302Image
on p.5302

Selected References

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

  1. Bagchi S., Raychaudhuri P., Nevins J. R. Phosphorylation-dependent activation of the adenovirus-inducible E2F transcription factor in a cell-free system. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4352–4356. doi: 10.1073/pnas.86.12.4352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bruner M., Dalie B., Spangler R., Harter M. L. Purification and biological characterization of an adenovirus type 2 E1A protein expressed in Escherichia coli. J Biol Chem. 1988 Mar 15;263(8):3984–3989. [PubMed] [Google Scholar]
  3. Carey M. F., Gerrard S. P., Cozzarelli N. R. Analysis of RNA polymerase III transcription complexes by gel filtration. J Biol Chem. 1986 Mar 25;261(9):4309–4317. [PubMed] [Google Scholar]
  4. Chatterjee P. K., Bruner M., Flint S. J., Harter M. L. DNA-binding properties of an adenovirus 289R E1A protein. EMBO J. 1988 Mar;7(3):835–841. doi: 10.1002/j.1460-2075.1988.tb02882.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cromlish J. A., Roeder R. G. Human transcription factor IIIC (TFIIIC). Purification, polypeptide structure, and the involvement of thiol groups in specific DNA binding. J Biol Chem. 1989 Oct 25;264(30):18100–18109. [PubMed] [Google Scholar]
  6. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Flint J., Shenk T. Adenovirus E1A protein paradigm viral transactivator. Annu Rev Genet. 1989;23:141–161. doi: 10.1146/annurev.ge.23.120189.001041. [DOI] [PubMed] [Google Scholar]
  8. Fowlkes D. M., Shenk T. Transcriptional control regions of the adenovirus VAI RNA gene. Cell. 1980 Nov;22(2 Pt 2):405–413. doi: 10.1016/0092-8674(80)90351-7. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Fuhrman S. A., Engelke D. R., Geiduschek E. P. HeLa cell RNA polymerase III transcription factors. Functional characterization of a fraction identified by its activity in a second template rescue assay. J Biol Chem. 1984 Feb 10;259(3):1934–1943. [PubMed] [Google Scholar]
  11. Gaynor R. B., Feldman L. T., Berk A. J. Transcription of class III genes activated by viral immediate early proteins. Science. 1985 Oct 25;230(4724):447–450. doi: 10.1126/science.2996135. [DOI] [PubMed] [Google Scholar]
  12. Gerster T., Roeder R. G. A herpesvirus trans-activating protein interacts with transcription factor OTF-1 and other cellular proteins. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6347–6351. doi: 10.1073/pnas.85.17.6347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gingeras T. R., Sciaky D., Gelinas R. E., Bing-Dong J., Yen C. E., Kelly M. M., Bullock P. A., Parsons B. L., O'Neill K. E., Roberts R. J. Nucleotide sequences from the adenovirus-2 genome. J Biol Chem. 1982 Nov 25;257(22):13475–13491. [PubMed] [Google Scholar]
  14. Green M. R., Treisman R., Maniatis T. Transcriptional activation of cloned human beta-globin genes by viral immediate-early gene products. Cell. 1983 Nov;35(1):137–148. doi: 10.1016/0092-8674(83)90216-7. [DOI] [PubMed] [Google Scholar]
  15. Green M., Loewenstein P. M., Pusztai R., Symington J. S. An adenovirus E1A protein domain activates transcription in vivo and in vitro in the absence of protein synthesis. Cell. 1988 Jun 17;53(6):921–926. doi: 10.1016/s0092-8674(88)90429-1. [DOI] [PubMed] [Google Scholar]
  16. Hai T. W., Horikoshi M., Roeder R. G., Green M. R. Analysis of the role of the transcription factor ATF in the assembly of a functional preinitiation complex. Cell. 1988 Sep 23;54(7):1043–1051. doi: 10.1016/0092-8674(88)90119-5. [DOI] [PubMed] [Google Scholar]
  17. Hoeffler W. K., Kovelman R., Roeder R. G. Activation of transcription factor IIIC by the adenovirus E1A protein. Cell. 1988 Jun 17;53(6):907–920. doi: 10.1016/s0092-8674(88)90409-6. [DOI] [PubMed] [Google Scholar]
  18. Hoeffler W. K., Roeder R. G. Enhancement of RNA polymerase III transcription by the E1A gene product of adenovirus. Cell. 1985 Jul;41(3):955–963. doi: 10.1016/s0092-8674(85)80076-3. [DOI] [PubMed] [Google Scholar]
  19. Imperiale M. J., Hart R. P., Nevins J. R. An enhancer-like element in the adenovirus E2 promoter contains sequences essential for uninduced and E1A-induced transcription. Proc Natl Acad Sci U S A. 1985 Jan;82(2):381–385. doi: 10.1073/pnas.82.2.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ko J. L., Dalie B. L., Goldman E., Harter M. L. Adenovirus-2 early region IA protein synthesized in Escherichia coli extracts indirectly associates with DNA. EMBO J. 1986 Jul;5(7):1645–1651. doi: 10.1002/j.1460-2075.1986.tb04407.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ko J. L., Harter M. L. Plasmid-directed synthesis of genuine adenovirus 2 early-region 1A and 1B proteins in Escherichia coli. Mol Cell Biol. 1984 Aug;4(8):1427–1439. doi: 10.1128/mcb.4.8.1427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kovelman R., Roeder R. G. Sarkosyl defines three intermediate steps in transcription initiation by RNA polymerase III: application to stimulation of transcription by E1A. Genes Dev. 1990 Apr;4(4):646–658. doi: 10.1101/gad.4.4.646. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Lee K. A., Green M. R. A cellular transcription factor E4F1 interacts with an E1a-inducible enhancer and mediates constitutive enhancer function in vitro. EMBO J. 1987 May;6(5):1345–1353. doi: 10.1002/j.1460-2075.1987.tb02374.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Leff T., Corden J., Elkaim R., Sassone-Corsi P. Transcriptional analysis of the adenovirus-5 EIII promoter: absence of sequence specificity for stimulation by EIa gene products. Nucleic Acids Res. 1985 Feb 25;13(4):1209–1221. doi: 10.1093/nar/13.4.1209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Leong K., Brunet L., Berk A. J. Factors responsible for the higher transcriptional activity of extracts of adenovirus-infected cells fractionate with the TATA box transcription factor. Mol Cell Biol. 1988 Apr;8(4):1765–1774. doi: 10.1128/mcb.8.4.1765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lillie J. W., Green M. R. Transcription activation by the adenovirus E1a protein. Nature. 1989 Mar 2;338(6210):39–44. doi: 10.1038/338039a0. [DOI] [PubMed] [Google Scholar]
  28. Liu F., Green M. R. A specific member of the ATF transcription factor family can mediate transcription activation by the adenovirus E1a protein. Cell. 1990 Jun 29;61(7):1217–1224. doi: 10.1016/0092-8674(90)90686-9. [DOI] [PubMed] [Google Scholar]
  29. Martin K. J., Lillie J. W., Green M. R. Evidence for interaction of different eukaryotic transcriptional activators with distinct cellular targets. Nature. 1990 Jul 12;346(6280):147–152. doi: 10.1038/346147a0. [DOI] [PubMed] [Google Scholar]
  30. Moran E., Zerler B., Harrison T. M., Mathews M. B. Identification of separate domains in the adenovirus E1A gene for immortalization activity and the activation of virus early genes. Mol Cell Biol. 1986 Oct;6(10):3470–3480. doi: 10.1128/mcb.6.10.3470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Murthy S. C., Bhat G. P., Thimmappaya B. Adenovirus EIIA early promoter: transcriptional control elements and induction by the viral pre-early EIA gene, which appears to be sequence independent. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2230–2234. doi: 10.1073/pnas.82.8.2230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Raychaudhuri P., Bagchi S., Nevins J. R. DNA-binding activity of the adenovirus-induced E4F transcription factor is regulated by phosphorylation. Genes Dev. 1989 May;3(5):620–627. doi: 10.1101/gad.3.5.620. [DOI] [PubMed] [Google Scholar]
  33. Simon M. C., Fisch T. M., Benecke B. J., Nevins J. R., Heintz N. Definition of multiple, functionally distinct TATA elements, one of which is a target in the hsp70 promoter for E1A regulation. Cell. 1988 Mar 11;52(5):723–729. doi: 10.1016/0092-8674(88)90410-2. [DOI] [PubMed] [Google Scholar]
  34. SivaRaman L., Thimmappaya B. Two promoter-specific host factors interact with adjacent sequences in an EIA-inducible adenovirus promoter. Proc Natl Acad Sci U S A. 1987 Sep;84(17):6112–6116. doi: 10.1073/pnas.84.17.6112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Spangler R., Bruner M., Dalie B., Harter M. L. Activation of adenovirus promoters by the adenovirus E1A protein in cell-free extracts. Science. 1987 Aug 28;237(4818):1044–1046. doi: 10.1126/science.2956686. [DOI] [PubMed] [Google Scholar]
  36. Weinmann R., Jaehning J. A., Raskas H. J., Roeder R. G. Viral RNA synthesis and levels of DNA-dependent RNA polymerases during replication of adenovirus 2. J Virol. 1975 Jan;17(1):114–126. doi: 10.1128/jvi.17.1.114-126.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yoshinaga S. K., Boulanger P. A., Berk A. J. Resolution of human transcription factor TFIIIC into two functional components. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3585–3589. doi: 10.1073/pnas.84.11.3585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yoshinaga S., Dean N., Han M., Berk A. J. Adenovirus stimulation of transcription by RNA polymerase III: evidence for an E1A-dependent increase in transcription factor IIIC concentration. EMBO J. 1986 Feb;5(2):343–354. doi: 10.1002/j.1460-2075.1986.tb04218.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES