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. 1983 Mar;80(5):1251–1255. doi: 10.1073/pnas.80.5.1251

Promoter-proximal pausing by RNA polymerase II in vitro: transcripts shorter than 20 nucleotides are not capped.

J A Coppola, A S Field, D S Luse
PMCID: PMC393573  PMID: 6572384

Abstract

We have synthesized RNA from cloned adenovirus 2 late promoter DNA in an in vitro transcription extract under UTP-limiting conditions. Under these circumstances, almost all of the alpha-amanitin-sensitive RNA produced is shorter than 20 nucleotides; most of these short transcripts are present in four species, 6, 7, 13, and 17 nucleotides. These short RNAs are initiated at the adenovirus 2 promoter, as judged by partial sequence analysis and by the abolition of their synthesis upon cleavage of the template DNA at sites which also abolish the production of full-length transcripts. All of the short transcripts can be chased, with excess UTP, into 197-base run-off transcripts; thus, these RNAs are precursors of full-length transcripts and not synthetic "dead ends." Significantly, none of these short RNAs is capped or 2'-O-methylated. However, 79-base run-off transcripts synthesized from this promoter with nonlimiting NTP levels are fully capped.

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Selected References

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

  1. Babich A., Nevins J. R., Darnell J. E., Jr Early capping of transcripts from the adenovirus major late transcription unit. Nature. 1980 Sep 18;287(5779):246–248. doi: 10.1038/287246a0. [DOI] [PubMed] [Google Scholar]
  2. Baker C. C., Ziff E. B. Biogenesis, structures, and sites of encoding of the 5' termini of adenovirus-2 mRNAs. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):415–428. doi: 10.1101/sqb.1980.044.01.045. [DOI] [PubMed] [Google Scholar]
  3. Breathnach R., Chambon P. Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem. 1981;50:349–383. doi: 10.1146/annurev.bi.50.070181.002025. [DOI] [PubMed] [Google Scholar]
  4. Bunick D., Zandomeni R., Ackerman S., Weinmann R. Mechanism of RNA polymerase II--specific initiation of transcription in vitro: ATP requirement and uncapped runoff transcripts. Cell. 1982 Jul;29(3):877–886. doi: 10.1016/0092-8674(82)90449-4. [DOI] [PubMed] [Google Scholar]
  5. Darnell J. E., Jr Variety in the level of gene control in eukaryotic cells. Nature. 1982 Jun 3;297(5865):365–371. doi: 10.1038/297365a0. [DOI] [PubMed] [Google Scholar]
  6. Fraser N. W., Sehgal P. B., Darnell J. E., Jr Multiple discrete sites for premature RNA chain termination late in adenovirus-2 infection: enhancement by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2571–2575. doi: 10.1073/pnas.76.6.2571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Lescure B., Bennetzen J., Sentenac A. In vitro transcription of the yeast alcohol dehydrogenase I gene by homologous RNA polymerase B (II). Selective initiation and discontinuous elongation on a supercoiled template. J Biol Chem. 1981 Nov 10;256(21):11018–11024. [PubMed] [Google Scholar]
  8. Lescure B., Williamson V., Sentenac A. Efficient and selective initiation by yeast RNA polymerase B in a dinucleotide-primed reaction. Nucleic Acids Res. 1981 Jan 10;9(1):31–45. doi: 10.1093/nar/9.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Manley J. L., Fire A., Cano A., Sharp P. A., Gefter M. L. DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3855–3859. doi: 10.1073/pnas.77.7.3855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Matsui T., Segall J., Weil P. A., Roeder R. G. Multiple factors required for accurate initiation of transcription by purified RNA polymerase II. J Biol Chem. 1980 Dec 25;255(24):11992–11996. [PubMed] [Google Scholar]
  11. McClure W. R., Cech C. L., Johnston D. E. A steady state assay for the RNA polymerase initiation reaction. J Biol Chem. 1978 Dec 25;253(24):8941–8948. [PubMed] [Google Scholar]
  12. Mizumoto K., Lipmann F. Transmethylation and transguanylylation in 5'-RNA capping system isolated from rat liver nuclei. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4961–4965. doi: 10.1073/pnas.76.10.4961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Salditt-Georgieff M., Harpold M., Chen-Kiang S., Darnell J. E., Jr The addition of 5' cap structures occurs early in hnRNA synthesis and prematurely terminated molecules are capped. Cell. 1980 Jan;19(1):69–78. doi: 10.1016/0092-8674(80)90389-x. [DOI] [PubMed] [Google Scholar]
  14. Tamm I., Kikuchi T., Darnell J. E., Jr, Salditt-Georgieff M. Short capped hnRNA precursor chains in HeLa cells: continued synthesis in the presence of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. Biochemistry. 1980 Jun 10;19(12):2743–2748. doi: 10.1021/bi00553a032. [DOI] [PubMed] [Google Scholar]
  15. Venkatesan S., Gershowitz A., Moss B. Purification and characterization of mRNA guanylyltransferase from HeLa cell nuclei. J Biol Chem. 1980 Apr 10;255(7):2829–2834. [PubMed] [Google Scholar]
  16. Venkatesan S., Moss B. Donor and acceptor specificities of HeLa cell mRNA guanylyltransferase. J Biol Chem. 1980 Apr 10;255(7):2835–2842. [PubMed] [Google Scholar]
  17. Volckaert G., Fiers W. A micromethod for base analysis of 32P-labeled oligoribonulcleotides. Anal Biochem. 1977 Nov;83(1):222–227. doi: 10.1016/0003-2697(77)90530-9. [DOI] [PubMed] [Google Scholar]
  18. Volckaert G., Fiers W. Micro thin-layer techniques for rapid sequence analysis of 32P-labeled RNA: double digestion and pancreatic ribonuclease analyses. Anal Biochem. 1977 Nov;83(1):228–239. doi: 10.1016/0003-2697(77)90531-0. [DOI] [PubMed] [Google Scholar]
  19. Weil P. A., Luse D. S., Segall J., Roeder R. G. Selective and accurate initiation of transcription at the Ad2 major late promotor in a soluble system dependent on purified RNA polymerase II and DNA. Cell. 1979 Oct;18(2):469–484. doi: 10.1016/0092-8674(79)90065-5. [DOI] [PubMed] [Google Scholar]
  20. Ziff E. B., Evans R. M. Coincidence of the promoter and capped 5' terminus of RNA from the adenovirus 2 major late transcription unit. Cell. 1978 Dec;15(4):1463–1475. doi: 10.1016/0092-8674(78)90070-3. [DOI] [PubMed] [Google Scholar]

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