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. 1991 Apr 25;19(8):1783–1790. doi: 10.1093/nar/19.8.1783

Elements involved in an in vitro block to transcription elongation at the end of the L1 mRNA family of adenovirus 2.

O Resnekov 1, R Pruzan 1, Y Aloni 1
PMCID: PMC328105  PMID: 1709491

Abstract

Using the 3' end of the L1 mRNA family of adenovirus 2 (Ad2) as a model system, we investigated transcription elongation following a poly(A) signal in a cell-free system. The results show that RNA polymerase II can halt transcription elongation at a T-rich stretch in the non-coding DNA strand 20 nucleotides downstream of the poly(A) signal. The block to transcription elongation is enhanced when Sarkosyl is included in the elongation reaction. Deletion studies narrowed the region which directs the elongation block at the T-rich stretch, to an upstream fragment of 53 nucleotides that is very dA-rich and also contains a functional poly(A) signal. The deletion studies and analysis by site-directed mutagenesis indicate that in the present system, RNA secondary structure, the stretch of T's and the poly(A) signal are not the dominant elements responsible for the elongation block. The block to transcription elongation at the T-rich stretch was also shown to be 5 times more effective in an uninfected extract than in an Ad2 infected extract, which is reminiscent of the in vivo situation and is consistent with the suggestion that a trans-acting factor is involved in modulating the elongation block at the T-rich stretch.

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Selected References

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

  1. Akusjärvi G., Persson H. Controls of RNA splicing and termination in the major late adenovirus transcription unit. Nature. 1981 Jul 30;292(5822):420–426. doi: 10.1038/292420a0. [DOI] [PubMed] [Google Scholar]
  2. Aloni Y., Hay N. Attenuation may regulate gene expression in animal viruses and cells. CRC Crit Rev Biochem. 1985;18(4):327–383. doi: 10.3109/10409238509086785. [DOI] [PubMed] [Google Scholar]
  3. Baek K. H., Sato K., Ito R., Agarwal K. RNA polymerase II transcription terminates at a specific DNA sequence in a HeLa cell-free reaction. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7623–7627. doi: 10.1073/pnas.83.20.7623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bengal E., Aloni Y. A block of transcription elongation by RNA polymerase II at synthetic sites in vitro. J Biol Chem. 1989 Jun 15;264(17):9791–9798. [PubMed] [Google Scholar]
  5. Bengal E., Flores O., Krauskopf A., Reinberg D., Aloni Y. Role of the mammalian transcription factors IIF, IIS, and IIX during elongation by RNA polymerase II. Mol Cell Biol. 1991 Mar;11(3):1195–1206. doi: 10.1128/mcb.11.3.1195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Birnstiel M. L., Busslinger M., Strub K. Transcription termination and 3' processing: the end is in site! Cell. 1985 Jun;41(2):349–359. doi: 10.1016/s0092-8674(85)80007-6. [DOI] [PubMed] [Google Scholar]
  7. Boles T. C., Hogan M. E. DNA structure equilibria in the human c-myc gene. Biochemistry. 1987 Jan 27;26(2):367–376. doi: 10.1021/bi00376a006. [DOI] [PubMed] [Google Scholar]
  8. Chow L. T., Broker T. R., Lewis J. B. Complex splicing patterns of RNAs from the early regions of adenovirus-2. J Mol Biol. 1979 Oct 25;134(2):265–303. doi: 10.1016/0022-2836(79)90036-6. [DOI] [PubMed] [Google Scholar]
  9. Connelly S., Manley J. L. A functional mRNA polyadenylation signal is required for transcription termination by RNA polymerase II. Genes Dev. 1988 Apr;2(4):440–452. doi: 10.1101/gad.2.4.440. [DOI] [PubMed] [Google Scholar]
  10. Davis T. L., Firulli A. B., Kinniburgh A. J. Ribonucleoprotein and protein factors bind to an H-DNA-forming c-myc DNA element: possible regulators of the c-myc gene. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9682–9686. doi: 10.1073/pnas.86.24.9682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Falck-Pedersen E., Logan J. Regulation of poly(A) site selection in adenovirus. J Virol. 1989 Feb;63(2):532–541. doi: 10.1128/jvi.63.2.532-541.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Galli G., Guise J. W., McDevitt M. A., Tucker P. W., Nevins J. R. Relative position and strengths of poly(A) sites as well as transcription termination are critical to membrane versus secreted mu-chain expression during B-cell development. Genes Dev. 1987 Jul;1(5):471–481. doi: 10.1101/gad.1.5.471. [DOI] [PubMed] [Google Scholar]
  13. Hales K. H., Birk J. M., Imperiale M. J. Analysis of adenovirus type 2 L1 RNA 3'-end formation in vivo and in vitro. J Virol. 1988 Apr;62(4):1464–1468. doi: 10.1128/jvi.62.4.1464-1468.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hawley D. K., Roeder R. G. Separation and partial characterization of three functional steps in transcription initiation by human RNA polymerase II. J Biol Chem. 1985 Jul 5;260(13):8163–8172. [PubMed] [Google Scholar]
  15. Iwamoto S., Eggerding F., Falck-Pederson E., Darnell J. E., Jr Transcription unit mapping in adenovirus: regions of termination. J Virol. 1986 Jul;59(1):112–119. doi: 10.1128/jvi.59.1.112-119.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kessler M., Ben-Asher E., Aloni Y. Elements modulating the block of transcription elongation at the adenovirus 2 attenuation site. J Biol Chem. 1989 Jun 15;264(17):9785–9790. [PubMed] [Google Scholar]
  17. Kitchingman G. R., Westphal H. The structure of adenovirus 2 early nuclear and cytoplasmic RNAs. J Mol Biol. 1980 Feb 15;137(1):23–48. doi: 10.1016/0022-2836(80)90155-2. [DOI] [PubMed] [Google Scholar]
  18. Koo H. S., Wu H. M., Crothers D. M. DNA bending at adenine . thymine tracts. Nature. 1986 Apr 10;320(6062):501–506. doi: 10.1038/320501a0. [DOI] [PubMed] [Google Scholar]
  19. Lanoix J., Acheson N. H. A rabbit beta-globin polyadenylation signal directs efficient termination of transcription of polyomavirus DNA. EMBO J. 1988 Aug;7(8):2515–2522. doi: 10.1002/j.1460-2075.1988.tb03099.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Leong K., Berk A. J. Adenovirus early region 1A protein increases the number of template molecules transcribed in cell-free extracts. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5844–5848. doi: 10.1073/pnas.83.16.5844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Logan J., Falck-Pedersen E., Darnell J. E., Jr, Shenk T. A poly(A) addition site and a downstream termination region are required for efficient cessation of transcription by RNA polymerase II in the mouse beta maj-globin gene. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8306–8310. doi: 10.1073/pnas.84.23.8306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Manley J. L., Fire A., Samuels M., Sharp P. A. In vitro transcription: whole-cell extract. Methods Enzymol. 1983;101:568–582. doi: 10.1016/0076-6879(83)01038-1. [DOI] [PubMed] [Google Scholar]
  23. Manley J. L., Sharp P. A., Gefter M. L. Rna synthesis in isolated nuclei processing of adenovirus serotype 2 late messenger rna precursors. J Mol Biol. 1982 Aug 25;159(4):581–599. doi: 10.1016/0022-2836(82)90102-4. [DOI] [PubMed] [Google Scholar]
  24. Nevins J. R., Wilson M. C. Regulation of adenovirus-2 gene expression at the level of transcriptional termination and RNA processing. Nature. 1981 Mar 12;290(5802):113–118. doi: 10.1038/290113a0. [DOI] [PubMed] [Google Scholar]
  25. Pfeiffer P., Hay N., Pruzan R., Jakobovits E. B., Aloni Y. In vitro premature termination in SV40 late transcription. EMBO J. 1983;2(2):185–191. doi: 10.1002/j.1460-2075.1983.tb01403.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Resnekov O., Aloni Y. RNA polymerase II is capable of pausing and prematurely terminating transcription at a precise location in vivo and in vitro. Proc Natl Acad Sci U S A. 1989 Jan;86(1):12–16. doi: 10.1073/pnas.86.1.12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Resnekov O., Ben-Asher E., Bengal E., Choder M., Hay N., Kessler M., Ragimov N., Seiberg M., Skolnik-David H., Aloni Y. Transcription termination in animal viruses and cells. Gene. 1988 Dec 10;72(1-2):91–104. doi: 10.1016/0378-1119(88)90130-8. [DOI] [PubMed] [Google Scholar]
  28. Resnekov O., Kessler M., Aloni Y. RNA secondary structure is an integral part of the in vitro mechanism of attenuation in simian virus 40. J Biol Chem. 1989 Jun 15;264(17):9953–9959. [PubMed] [Google Scholar]
  29. Sato K., Ito R., Baek K. H., Agarwal K. A specific DNA sequence controls termination of transcription in the gastrin gene. Mol Cell Biol. 1986 Apr;6(4):1032–1043. doi: 10.1128/mcb.6.4.1032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Seiberg M., Kessler M., Levine A. J., Aloni Y. Human RNA polymerase II can prematurely terminate transcription of the adenovirus type 2 late transcription unit at a precise site that resembles a prokaryotic termination signal. Virus Genes. 1987 Nov;1(1):97–116. doi: 10.1007/BF00125689. [DOI] [PubMed] [Google Scholar]
  31. Shaw A. R., Ziff E. B. Transcripts from the adenovirus-2 major late promoter yield a single early family of 3' coterminal mRNAs and five late families. Cell. 1980 Dec;22(3):905–916. doi: 10.1016/0092-8674(80)90568-1. [DOI] [PubMed] [Google Scholar]
  32. Thomas G. P., Mathews M. B. DNA replication and the early to late transition in adenovirus infection. Cell. 1980 Nov;22(2 Pt 2):523–533. doi: 10.1016/0092-8674(80)90362-1. [DOI] [PubMed] [Google Scholar]
  33. Tinoco I., Jr, Borer P. N., Dengler B., Levin M. D., Uhlenbeck O. C., Crothers D. M., Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. doi: 10.1038/newbio246040a0. [DOI] [PubMed] [Google Scholar]
  34. Whitelaw E., Proudfoot N. Alpha-thalassaemia caused by a poly(A) site mutation reveals that transcriptional termination is linked to 3' end processing in the human alpha 2 globin gene. EMBO J. 1986 Nov;5(11):2915–2922. doi: 10.1002/j.1460-2075.1986.tb04587.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wickens M., Stephenson P. Role of the conserved AAUAAA sequence: four AAUAAA point mutants prevent messenger RNA 3' end formation. Science. 1984 Nov 30;226(4678):1045–1051. doi: 10.1126/science.6208611. [DOI] [PubMed] [Google Scholar]
  36. Wilusz J., Shenk T. A 64 kd nuclear protein binds to RNA segments that include the AAUAAA polyadenylation motif. Cell. 1988 Jan 29;52(2):221–228. doi: 10.1016/0092-8674(88)90510-7. [DOI] [PubMed] [Google Scholar]

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