Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1993 Jul 25;21(15):3451–3457. doi: 10.1093/nar/21.15.3451

Efficient expression of a protein coding gene under the control of an RNA polymerase I promoter.

T D Palmer 1, A D Miller 1, R H Reeder 1, B McStay 1
PMCID: PMC331444  PMID: 8393988

Abstract

In mammalian cells, RNA polymerase I transcripts are uncapped and retain a polyphosphate 5' terminus. It is probably for this reason that they are poorly translated as messenger RNA. We show in this report that insertion of an Internal Ribosome Entry Site (IRES) into the 5' leader of an RNA polymerase I transcript overcomes the block to translation, presumably by substituting for the 5' trimethyl G cap. Addition of an SV40 polyA addition signal also enhances protein production from the RNA polymerase I transcript. RNA Polymerase I driven expression vectors containing both elements produce protein at levels comparable to that produced from RNA polymerase II driven expression vectors which utilize a retroviral LTR. RNA Polymerase I driven expression vectors may have a variety of uses both for basic research and for practical expression of recombinant proteins.

Full text

PDF
3451

Selected References

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

  1. Bell S. P., Jantzen H. M., Tjian R. Assembly of alternative multiprotein complexes directs rRNA promoter selectivity. Genes Dev. 1990 Jun;4(6):943–954. doi: 10.1101/gad.4.6.943. [DOI] [PubMed] [Google Scholar]
  2. Bell S. P., Pikaard C. S., Reeder R. H., Tjian R. Molecular mechanisms governing species-specific transcription of ribosomal RNA. Cell. 1989 Nov 3;59(3):489–497. doi: 10.1016/0092-8674(89)90032-9. [DOI] [PubMed] [Google Scholar]
  3. Corsaro C. M., Pearson M. L. Enhancing the efficiency of DNA-mediated gene transfer in mammalian cells. Somatic Cell Genet. 1981 Sep;7(5):603–616. doi: 10.1007/BF01549662. [DOI] [PubMed] [Google Scholar]
  4. Elroy-Stein O., Fuerst T. R., Moss B. Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6126–6130. doi: 10.1073/pnas.86.16.6126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fleischer S., Grummt I. Expression of an mRNA coding gene under the control of an RNA polymerase I promoter. EMBO J. 1983;2(12):2319–2322. doi: 10.1002/j.1460-2075.1983.tb01740.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Grummt I., Skinner J. A. Efficient transcription of a protein-coding gene from the RNA polymerase I promoter in transfected cells. Proc Natl Acad Sci U S A. 1985 Feb;82(3):722–726. doi: 10.1073/pnas.82.3.722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Jang S. K., Davies M. V., Kaufman R. J., Wimmer E. Initiation of protein synthesis by internal entry of ribosomes into the 5' nontranslated region of encephalomyocarditis virus RNA in vivo. J Virol. 1989 Apr;63(4):1651–1660. doi: 10.1128/jvi.63.4.1651-1660.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kuehn M., Arnheim N. Nucleotide sequence of the genetically labile repeated elements 5' to the origin of mouse rRNA transcription. Nucleic Acids Res. 1983 Jan 11;11(1):211–224. doi: 10.1093/nar/11.1.211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Learned R. M., Tjian R. In vitro transcription of human ribosomal RNA genes by RNA polymerase I. J Mol Appl Genet. 1982;1(6):575–584. [PubMed] [Google Scholar]
  10. Lopata M. A., Cleveland D. W., Sollner-Webb B. RNA polymerase specificity of mRNA production and enhancer action. Proc Natl Acad Sci U S A. 1986 Sep;83(18):6677–6681. doi: 10.1073/pnas.83.18.6677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Mahajan P. B., Gokal P. K., Thompson E. A. Hormonal regulation of transcription of rDNA. The role of TFIC in formation of initiation complexes. J Biol Chem. 1990 Sep 25;265(27):16244–16247. [PubMed] [Google Scholar]
  12. Miller A. D., Jolly D. J., Friedmann T., Verma I. M. A transmissible retrovirus expressing human hypoxanthine phosphoribosyltransferase (HPRT): gene transfer into cells obtained from humans deficient in HPRT. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4709–4713. doi: 10.1073/pnas.80.15.4709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Miller A. D., Rosman G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980-2, 984-6, 989-90. [PMC free article] [PubMed] [Google Scholar]
  14. Nogi Y., Yano R., Nomura M. Synthesis of large rRNAs by RNA polymerase II in mutants of Saccharomyces cerevisiae defective in RNA polymerase I. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3962–3966. doi: 10.1073/pnas.88.9.3962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Quade K. Transformation of mammalian cells by avian myelocytomatosis virus and avian erythroblastosis virus. Virology. 1979 Oct 30;98(2):461–465. doi: 10.1016/0042-6822(79)90569-5. [DOI] [PubMed] [Google Scholar]
  16. Reeder R. H., Sollner-Webb B., Wahn H. L. Sites of transcription initiation in vivo on Xenopus laevis ribosomal DNA. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5402–5406. doi: 10.1073/pnas.74.12.5402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Rudenko G., Chung H. M., Pham V. P., Van der Ploeg L. H. RNA polymerase I can mediate expression of CAT and neo protein-coding genes in Trypanosoma brucei. EMBO J. 1991 Nov;10(11):3387–3397. doi: 10.1002/j.1460-2075.1991.tb04903.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Smale S. T., Tjian R. Transcription of herpes simplex virus tk sequences under the control of wild-type and mutant human RNA polymerase I promoters. Mol Cell Biol. 1985 Feb;5(2):352–362. doi: 10.1128/mcb.5.2.352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sonenberg N. Poliovirus translation. Curr Top Microbiol Immunol. 1990;161:23–47. doi: 10.1007/978-3-642-75602-3_2. [DOI] [PubMed] [Google Scholar]
  20. Surmacz E., Kaczmarek L., Rønning O., Baserga R. Activation of the ribosomal DNA promoter in cells exposed to insulinlike growth factor I. Mol Cell Biol. 1987 Feb;7(2):657–663. doi: 10.1128/mcb.7.2.657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Surmacz E., Rønning O., Kaczmarek L., Baserga R. Regulation of the expression of the SV40 T-antigen coding gene under the control of an rDNA promoter. J Cell Physiol. 1986 Jun;127(3):357–365. doi: 10.1002/jcp.1041270303. [DOI] [PubMed] [Google Scholar]
  22. Zomerdijk J. C., Kieft R., Borst P. Efficient production of functional mRNA mediated by RNA polymerase I in Trypanosoma brucei. Nature. 1991 Oct 24;353(6346):772–775. doi: 10.1038/353772a0. [DOI] [PubMed] [Google Scholar]
  23. Zomerdijk J. C., Kieft R., Shiels P. G., Borst P. Alpha-amanitin-resistant transcription units in trypanosomes: a comparison of promoter sequences for a VSG gene expression site and for the ribosomal RNA genes. Nucleic Acids Res. 1991 Oct 11;19(19):5153–5158. doi: 10.1093/nar/19.19.5153. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES