Abstract
Two transcription factors, rat UBF (rUBF) and rat SL-1 are required for the efficient transcription of the rat promoter in vitro. In vitro studies have established that two broadly defined cis-acting domains, the core promoter element and the upstream promoter element, cooperate to direct correct transcription by RNA polymerase I. The ability of UBF to bind to two linker-scanning mutants of the upstream promoter element, which did not respond to the addition of UBF in in vitro transcription assays, was assessed by DNase foot-printing. UBF protected the same region of the promoter in the linker-scanning mutant in BSM 129/124 as it did in the wild-type, but did not yield a typical footprint over the promoter in the linker-scanning mutant BSM 106/101.
Previously we reported that promoters with mutant core promoters elements, either the guanine at −16 or −7 substituted by an adenine, were inactive in vitro unless the assays were supplemented with UBF. Those results suggested that the binding of UBF upstream of the core was required for the promotion of transcription. The interactions between the core and upstream promoter elements were assessed by constructing double mutants of the promoter. In two constructs the conserved guanines at either −16 or −7 were altered in a deletion mutant (−86) that did not respond to UBF. In a third construct the guanine at −16 in BSM 129/124 was changed to an adenine. These bidomain mutant constructs did not respond to the addition of UBF in an in vitro transcription assay, confirming that the rescue of the core promoter mutants requires an intact and functional upstream promoter element. These in vitro results have been further examined in vivo by transfecting various constructs into CHO cells. The in vivo studies indicate that besides the nucleotides mutated in BSM 129/124 and BSM 106/101, a third element in the UPE, circa nucleotides −117/−112, may also play a role in transcription.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Cassidy B, Haglund R, Rothblum L: Regions upstream from the core promoter of the rat ribosomal gene are required for the formation of a stable transcription initiation complex by RNA polymerase I in vitro. Biochim Biophys Acta 909: 113–144, 1987
Grummt I: Nucleotide sequence requirements for specific initiation of transcription by RNA polymerase I. Proc Natl Acad Sci USA 79: 6908–6911, 1982
Haltiner M, Smale S, Tjian R: Two distinct promoter elements in the human rRNA gene identified by linker scanning mutagenesis. Mol Cell Biol 6: 227–235, 1986
Learned R, Smale S, Haltiner M, Tjian R: Regulation of human ribosomal RNA transcription. Proc Natl Acad Sci USA 80: 3558–3562, 1983
Yamamoto O, Takakusa N, Mishima Y, Kominami R, Muramatsu M: Determination of the promoter region of the mouse ribosomal RNA gene by an in vitro transcription system. Proc Natl Acad Sci USA 81: 299–303, 1984
Miller K, Tower J, Sollner-Webb B: A complex control region of the mouse rRNA gene directs accurate initiation by RNA polymerase I. Mol Cell Biol 5: 554–562, 1985
Smale S, 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 5: 352–362, 1985
Grummt I, Kuhn A, Bartsch I, Rosenbauer H: A transcription terminator located upstream of the mouse rDNA initiation site affects rRNA synthesis. Cell 47: 901–911, 1986
Henderson S, Sollner-Webb B: A transcriptional terminator is a novel element of the promoter of the mouse ribosomal RNA gene. Cell 47: 891–900, 1986
McStay B, Reeder RH: A termination site for Xenopus RNA polymerase I also acts as an element of an adjacent promoter. Cell 47: 913–920, 1986
Tower J Culotta V, Sollner-Webb B: Factors and nucleotide sequences that direct ribosomal DNA transcription and their relationship to the stable transcription complex. Mol Cell Biol 6: 3451–3462, 1986
Breathnach R, Chambon P: Organization and expression of eukaryotic split genes coding for proteins. Ann Rev Biochem 50: 349–383, 1981
Culotta V, Wilkinson J, Sollner-Webb B: Mouse and Frog violate the paradigm of species-specific transcription of ribosomal RNA genes. Proc Natl Acad Sci USA 84: 7498–7502, 1987
Miesfeld R, Arnheim N: Species-specific rDNA transcription is due to promoter-specific binding factor. Mol Cell Biol 4: 21–227, 1984
Mishima Y, Financsek L, Kominami R, Muramatsu M: Fractionation and reconstitution of factors required for accurate transcription of mammalian ribosomal RNA genes: identification of a species-specific factor. Nucl Acids Res 10:6659–6670, 1982
Clos J, Buttgercit D, Grummt I: A purified transcription factor (TIF-1B) binds to essential sequences of the mouse rDNA promoter. Proc Natl Acad Sci USA 83: 604–608, 1986
Smith SD, Oriahi E, Lowe D, Yang-Yen H-F, O'Mahony D, Rose K, Chen K, Rothblum LI: Characterization of factors that direct transcription of rat ribosomal DNA. Mol Cell Biol 10: 3105–3116, 1990
Learned R, Cordes S, Tjian R: Purification and characterization of transcription factor that confers promoter specificity. Mol Cell Biol 5: 1358–1369, 1985
Grummt I, Roth E, Paule M: Ribosomal RNA transcription in vitro is species specific. Nature 296: 173–174, 1982
Bell S, Learned R, Jantzen H-M, Tjian R: Functional Cooperativity between transcription factors UBFI and SLl mediates human ribosomal RNA synthesis. Science 241: 1192–1197, 1988
Learned R, Learned T, Haltiner M, Tjian R: Human rRNA transcription is modulated by the coordinate binding of two factors to an upstream control element. Cell 45: 847–857, 1986
Sanger F, Nicklen S, Coulson A: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467, 1977
Haglund RE, Rothblum LI: Isolation, fractionation and reconstitution of a nuclear extract capable of transcribing ribosomal DNA. Mol Cell Biochem 73: 11–20, 1987
Smith SD, Oriahi E, Yang-Yen H-F, Xie WQ, Chen C, Rothblum LI: Interaction of RNA polymerase I transcription factors with a promoter in the nontranscribed spacer of rat ribosomal DNA. Nucl Acids Res 18: 1677–1685, 1990
Wigler M, Pellicer A, Silvestein S, Axel R: Biochemical transfer of single copy genes using total cellular DNA as donor. Cell 22: 725–731, 1987
Favoloro J, Triesman R, Kamen R: Transcription maps of polyoma virus-specific RNA: Analysis by two-dimensional nuclease Sl gel mapping. Methods in Enzym 65: 718–749, 1980
Zinn K, Maio D, Maniatis T: Identification of Two Distinct Regulatory Regions Adjacent to the Human R-Interferon Gene. Cell 34: 865–879, 1987
Pikaard C, Smith D, Reeder R, Rothblum L: rUBF, an RNA Polymerase I Transcription Factor from Rats, Produces DNase I Footprints Identical to Those Produced by xUBF, Its Homolog from Frogs. Mol Cell Biol 10: 3810–3812, 1990
Linn S, Riggs A: Photochemical attachment of lac repressor to bromodeoxymidine substituted lac operator by ultraviolet radiation. Proc Natl Acad Sci USA 71: 947–951, 1974
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Xie, W.Q., O'Mahony, D.J., Smith, S.D. et al. Complementary in vivo and in vitro analyses of the interactions between the cis-acting elements of the rat rDNA promoter. Mol Cell Biochem 104, 127–135 (1991). https://doi.org/10.1007/BF00229812
Issue Date:
DOI: https://doi.org/10.1007/BF00229812