Abstract
A 16,000-dalton, high-mobility-group-like (HMG-like) DNA-binding protein, referred to as p16, has been purified to homogeneity from Novikoff hepatoma ascites cells. p16 binds specifically to a portion of the 5' flanking region of the rat rRNA gene (-620 to -417), which is part of the upstream activator sequence identified previously (B. G. Cassidy, H.-F. Yang-Yen, and L. I. Rothblum, Mol. Cell. Biol. 6:2766-2773, 1986). p16 also binds to a segment of the external transcribed spacer (+352 to +545). In vitro reconstituted transcription experiments demonstrated that the addition of p16 stimulated rRNA synthesis up to ca. fourfold. The stimulation was dose dependent and saturable. The effect of p16 on ribosomal gene transcription was also dependent on the presence of either the upstream or the downstream DNA-binding site, or both. The amino acid composition of p16 is very similar to that of HMG-I, suggesting that p16 may be a member of the HMG-I family of proteins. In this case, our results suggest that HMG proteins may play an important role in the regulation of the rRNA gene expression.
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Selected References
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- Angel P., Imagawa M., Chiu R., Stein B., Imbra R. J., Rahmsdorf H. J., Jonat C., Herrlich P., Karin M. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell. 1987 Jun 19;49(6):729–739. doi: 10.1016/0092-8674(87)90611-8. [DOI] [PubMed] [Google Scholar]
- Birkenmeier E. H., Brown D. D., Jordan E. A nuclear extract of Xenopus laevis oocytes that accurately transcribes 5S RNA genes. Cell. 1978 Nov;15(3):1077–1086. doi: 10.1016/0092-8674(78)90291-x. [DOI] [PubMed] [Google Scholar]
- Cassidy B. G., Yang-Yen H. F., Rothblum L. I. Additional RNA polymerase I initiation site within the nontranscribed spacer region of the rat rRNA gene. Mol Cell Biol. 1987 Jul;7(7):2388–2396. doi: 10.1128/mcb.7.7.2388. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cassidy B. G., Yang-Yen H. F., Rothblum L. I. Transcriptional role for the nontranscribed spacer of rat ribosomal DNA. Mol Cell Biol. 1986 Aug;6(8):2766–2773. doi: 10.1128/mcb.6.8.2766. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cassidy B., Haglund R., Rothblum L. I. 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. 1987 Jul 14;909(2):133–144. doi: 10.1016/0167-4781(87)90035-2. [DOI] [PubMed] [Google Scholar]
- De Winter R. F., Moss T. Spacer promoters are essential for efficient enhancement of X. laevis ribosomal transcription. Cell. 1986 Jan 31;44(2):313–318. doi: 10.1016/0092-8674(86)90765-8. [DOI] [PubMed] [Google Scholar]
- Dixit A., Garg L. C., Chao W., Jacob S. T. An enhancer element in the far upstream spacer region of rat ribosomal RNA gene. J Biol Chem. 1987 Aug 25;262(24):11616–11622. [PubMed] [Google Scholar]
- Elion E. A., Warner J. R. An RNA polymerase I enhancer in Saccharomyces cerevisiae. Mol Cell Biol. 1986 Jun;6(6):2089–2097. doi: 10.1128/mcb.6.6.2089. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Elton T. S., Nissen M. S., Reeves R. Specific A . T DNA sequence binding of RP-HPLC purified HMG-I. Biochem Biophys Res Commun. 1987 Feb 27;143(1):260–265. doi: 10.1016/0006-291x(87)90659-0. [DOI] [PubMed] [Google Scholar]
- 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]
- Gillies S. D., Morrison S. L., Oi V. T., Tonegawa S. A tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene. Cell. 1983 Jul;33(3):717–728. doi: 10.1016/0092-8674(83)90014-4. [DOI] [PubMed] [Google Scholar]
- Goodwin G. H., Cockerill P. N., Kellam S., Wright C. A. Fractionation by high-performance liquid chromatography of the low-molecular-mass high-mobility-group (HMG) chromosomal proteins present in proliferating rat cells and an investigation of the HMG proteins present in virus transformed cells. Eur J Biochem. 1985 May 15;149(1):47–51. doi: 10.1111/j.1432-1033.1985.tb08891.x. [DOI] [PubMed] [Google Scholar]
- Grummt I., Kuhn A., Bartsch I., Rosenbauer H. A transcription terminator located upstream of the mouse rDNA initiation site affects rRNA synthesis. Cell. 1986 Dec 26;47(6):901–911. doi: 10.1016/0092-8674(86)90805-6. [DOI] [PubMed] [Google Scholar]
- Grummt I. Nucleotide sequence requirements for specific initiation of transcription by RNA polymerase I. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6908–6911. doi: 10.1073/pnas.79.22.6908. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Grummt I., Rosenbauer H., Niedermeyer I., Maier U., Ohrlein A. A repeated 18 bp sequence motif in the mouse rDNA spacer mediates binding of a nuclear factor and transcription termination. Cell. 1986 Jun 20;45(6):837–846. doi: 10.1016/0092-8674(86)90558-1. [DOI] [PubMed] [Google Scholar]
- Grummt I., Roth E., Paule M. R. Ribosomal RNA transcription in vitro is species specific. Nature. 1982 Mar 11;296(5853):173–174. doi: 10.1038/296173a0. [DOI] [PubMed] [Google Scholar]
- Grummt I. Specific transcription of mouse ribosomal DNA in a cell-free system that mimics control in vivo. Proc Natl Acad Sci U S A. 1981 Feb;78(2):727–731. doi: 10.1073/pnas.78.2.727. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hager D. A., Burgess R. R. Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem. 1980 Nov 15;109(1):76–86. doi: 10.1016/0003-2697(80)90013-5. [DOI] [PubMed] [Google Scholar]
- Haglund R. E., Rothblum L. I. Isolation, fractionation and reconstitution of a nuclear extract capable of transcribing ribosomal DNA. Mol Cell Biochem. 1987 Jan;73(1):11–20. doi: 10.1007/BF00229371. [DOI] [PubMed] [Google Scholar]
- Haltiner M. M., Smale S. T., Tjian R. Two distinct promoter elements in the human rRNA gene identified by linker scanning mutagenesis. Mol Cell Biol. 1986 Jan;6(1):227–235. doi: 10.1128/mcb.6.1.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Harrington C. A., Chikaraishi D. M. Transcription of spacer sequences flanking the rat 45S ribosomal DNA gene. Mol Cell Biol. 1987 Jan;7(1):314–325. doi: 10.1128/mcb.7.1.314. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Henderson S., Sollner-Webb B. A transcriptional terminator is a novel element of the promoter of the mouse ribosomal RNA gene. Cell. 1986 Dec 26;47(6):891–900. doi: 10.1016/0092-8674(86)90804-4. [DOI] [PubMed] [Google Scholar]
- Jackson J. B., Pollock J. M., Jr, Rill R. L. Chromatin fractionation procedure that yields nucleosomes containing near-stoichiometric amounts of high mobility group nonhistone chromosomal proteins. Biochemistry. 1979 Aug 21;18(17):3739–3748. doi: 10.1021/bi00584a015. [DOI] [PubMed] [Google Scholar]
- Kohorn B. D., Rae P. M. Localization of DNA sequences promoting RNA polymerase I activity in Drosophila. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3265–3268. doi: 10.1073/pnas.80.11.3265. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kunkel G. R., Martinson H. G. Nucleosomes will not form on double-stranded RNa or over poly(dA).poly(dT) tracts in recombinant DNA. Nucleic Acids Res. 1981 Dec 21;9(24):6869–6888. doi: 10.1093/nar/9.24.6869. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Labhart P., Reeder R. H. Enhancer-like properties of the 60/81 bp elements in the ribosomal gene spacer of Xenopus laevis. Cell. 1984 May;37(1):285–289. doi: 10.1016/0092-8674(84)90324-6. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Learned R. M., Smale S. T., Haltiner M. M., Tjian R. Regulation of human ribosomal RNA transcription. Proc Natl Acad Sci U S A. 1983 Jun;80(12):3558–3562. doi: 10.1073/pnas.80.12.3558. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Long E. O., Dawid I. B. Repeated genes in eukaryotes. Annu Rev Biochem. 1980;49:727–764. doi: 10.1146/annurev.bi.49.070180.003455. [DOI] [PubMed] [Google Scholar]
- Lund T., Dahl K. H., Mørk E., Holtlund J., Laland S. G. The human chromosomal protein HMG I contains two identical palindrome amino acid sequences. Biochem Biophys Res Commun. 1987 Jul 31;146(2):725–730. doi: 10.1016/0006-291x(87)90589-4. [DOI] [PubMed] [Google Scholar]
- Lund T., Holtlund J., Fredriksen M., Laland S. G. On the presence of two new high mobility group-like proteins in HeLa S3 cells. FEBS Lett. 1983 Feb 21;152(2):163–167. doi: 10.1016/0014-5793(83)80370-6. [DOI] [PubMed] [Google Scholar]
- Mandal R. K. The organization and transcription of eukaryotic ribosomal RNA genes. Prog Nucleic Acid Res Mol Biol. 1984;31:115–160. doi: 10.1016/s0079-6603(08)60376-1. [DOI] [PubMed] [Google Scholar]
- 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]
- Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
- McStay B., Reeder R. H. A termination site for Xenopus RNA polymerase I also acts as an element of an adjacent promoter. Cell. 1986 Dec 26;47(6):913–920. doi: 10.1016/0092-8674(86)90806-8. [DOI] [PubMed] [Google Scholar]
- Miesfeld R., Arnheim N. Species-specific rDNA transcription is due to promoter-specific binding factors. Mol Cell Biol. 1984 Feb;4(2):221–227. doi: 10.1128/mcb.4.2.221. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Miller K. G., Tower J., Sollner-Webb B. A complex control region of the mouse rRNA gene directs accurate initiation by RNA polymerase I. Mol Cell Biol. 1985 Mar;5(3):554–562. doi: 10.1128/mcb.5.3.554. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mishima Y., Financsek I., Kominami R., Muramatsu M. Fractionation and reconstitution of factors required for accurate transcription of mammalian ribosomal RNA genes: identification of a species-dependent initiation factor. Nucleic Acids Res. 1982 Nov 11;10(21):6659–6670. doi: 10.1093/nar/10.21.6659. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Moss T. A transcriptional function for the repetitive ribosomal spacer in Xenopus laevis. Nature. 1983 Mar 17;302(5905):223–228. doi: 10.1038/302223a0. [DOI] [PubMed] [Google Scholar]
- Mroczka D. L., Cassidy B., Busch H., Rothblum L. I. Characterization of rat ribosomal DNA. The highly repetitive sequences that flank the ribosomal RNA transcription unit are homologous and contain RNA polymerase III transcription initiation sites. J Mol Biol. 1984 Mar 25;174(1):141–162. doi: 10.1016/0022-2836(84)90369-3. [DOI] [PubMed] [Google Scholar]
- Prunell A. Nucleosome reconstitution on plasmid-inserted poly(dA) . poly(dT). EMBO J. 1982;1(2):173–179. doi: 10.1002/j.1460-2075.1982.tb01143.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Queen C., Baltimore D. Immunoglobulin gene transcription is activated by downstream sequence elements. Cell. 1983 Jul;33(3):741–748. doi: 10.1016/0092-8674(83)90016-8. [DOI] [PubMed] [Google Scholar]
- Reeder R. H., Roan J. G., Dunaway M. Spacer regulation of Xenopus ribosomal gene transcription: competition in oocytes. Cell. 1983 Dec;35(2 Pt 1):449–456. doi: 10.1016/0092-8674(83)90178-2. [DOI] [PubMed] [Google Scholar]
- Rothblum L. I., Reddy R., Cassidy B. Transcription initiation site of rat ribosomal DNA. Nucleic Acids Res. 1982 Nov 25;10(22):7345–7362. doi: 10.1093/nar/10.22.7345. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rungger D., Crippa M., Trendelenburg M. F., Scheer U., Franke W. W. Visualization of rDNA spacer transcription in Xenopus oocytes treated with fluorouridine. Exp Cell Res. 1978 Oct 15;116(2):481–486. doi: 10.1016/0014-4827(78)90476-7. [DOI] [PubMed] [Google Scholar]
- Sandeen G., Wood W. I., Felsenfeld G. The interaction of high mobility proteins HMG14 and 17 with nucleosomes. Nucleic Acids Res. 1980 Sep 11;8(17):3757–3778. doi: 10.1093/nar/8.17.3757. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schöler H. R., Gruss P. Cell type-specific transcriptional enhancement in vitro requires the presence of trans-acting factors. EMBO J. 1985 Nov;4(11):3005–3013. doi: 10.1002/j.1460-2075.1985.tb04036.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Simpson R. T., Künzler P. Cromatin and core particles formed from the inner histones and synthetic polydeoxyribonucleotides of defined sequence. Nucleic Acids Res. 1979 Apr;6(4):1387–1415. doi: 10.1093/nar/6.4.1387. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Solomon M. J., Strauss F., Varshavsky A. A mammalian high mobility group protein recognizes any stretch of six A.T base pairs in duplex DNA. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1276–1280. doi: 10.1073/pnas.83.5.1276. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Struhl K. Naturally occurring poly(dA-dT) sequences are upstream promoter elements for constitutive transcription in yeast. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8419–8423. doi: 10.1073/pnas.82.24.8419. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tower J., Culotta V. C., Sollner-Webb B. Factors and nucleotide sequences that direct ribosomal DNA transcription and their relationship to the stable transcription complex. Mol Cell Biol. 1986 Oct;6(10):3451–3462. doi: 10.1128/mcb.6.10.3451. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tremethick D. J., Molloy P. L. High mobility group proteins 1 and 2 stimulate transcription in vitro by RNA polymerases II and III. J Biol Chem. 1986 May 25;261(15):6986–6992. [PubMed] [Google Scholar]
- Vidali G., Boffa L. C., Allfrey V. G. Selective release of chromosomal proteins during limited DNAase 1 digestion of avian erythrocyte chromatin. Cell. 1977 Oct;12(2):409–415. doi: 10.1016/0092-8674(77)90117-9. [DOI] [PubMed] [Google Scholar]
- Wandelt C., Grummt I. Formation of stable preinitiation complexes is a prerequisite for ribosomal DNA transcription in vitro. Nucleic Acids Res. 1983 Jun 11;11(11):3795–3809. doi: 10.1093/nar/11.11.3795. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weil P. A., Segall J., Harris B., Ng S. Y., Roeder R. G. Faithful transcription of eukaryotic genes by RNA polymerase III in systems reconstituted with purified DNA templates. J Biol Chem. 1979 Jul 10;254(13):6163–6173. [PubMed] [Google Scholar]
- Weisbrod S., Groudine M., Weintraub H. Interaction of HMG 14 and 17 with actively transcribed genes. Cell. 1980 Jan;19(1):289–301. doi: 10.1016/0092-8674(80)90410-9. [DOI] [PubMed] [Google Scholar]
- Weisbrod S., Weintraub H. Isolation of actively transcribed nucleosomes using immobilized HMG 14 and 17 and an analysis of alpha-globin chromatin. Cell. 1981 Feb;23(2):391–400. doi: 10.1016/0092-8674(81)90134-3. [DOI] [PubMed] [Google Scholar]
- Wu H. M., Crothers D. M. The locus of sequence-directed and protein-induced DNA bending. Nature. 1984 Apr 5;308(5959):509–513. doi: 10.1038/308509a0. [DOI] [PubMed] [Google Scholar]
- Yamamoto O., Takakusa N., Mishima Y., Kominami R., Muramatsu M. Determination of the promoter region of mouse ribosomal RNA gene by an in vitro transcription system. Proc Natl Acad Sci U S A. 1984 Jan;81(2):299–303. doi: 10.1073/pnas.81.2.299. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yang-Yen H. F., Rothblum L. I. Partial nucleotide sequence of a 3.4 kb fragment from the rat ribosomal DNA nontranscribed spacer. Nucleic Acids Res. 1986 Jul 11;14(13):5557–5557. doi: 10.1093/nar/14.13.5557. [DOI] [PMC free article] [PubMed] [Google Scholar]