title: Efficient transcription by RNA polymerase I using recombinant core factor.
abstract: Transcription of ribosomal DNA by RNA polymerase I is a central feature of eukaryotic ribosome biogenesis. Since ribosome synthesis is closely linked to cell proliferation, there is a need to define the molecular mechanisms that control transcription by RNA polymerase I. To fully define the factors that control RNA polymerase I activity, biochemical analyses using purified transcription factors are essential. Although such assays exist, one limitation is the low abundance and difficult purification strategies required for some of the essential transcription factors for RNA polymerase I. Here, we describe a new method for expression and purification of the three subunit core factor complex from Escherichia coli. We demonstrate that the recombinant material is more active than yeast-derived core factor in assays for RNA polymerase I transcription in vitro. Finally, we use recombinant core factor to differentiate between two opposing models for the role of the TATA-binding protein in transcription by RNA polymerase I.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Alabama-Birmingham, 720 20th Street South, Birmingham, AL 35294, USA.
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Bedwell
authors.forename: Francis D
authors.initials: FD
authors.lastname: Appling
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Anderson
authors.forename: David A
authors.initials: DA
authors.lastname: Schneider
mesh_terms: D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D035463:Pol1 Transcription Initiation Complex Proteins / Q000235:genetics*; D012318:RNA Polymerase I / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D035463:Pol1 Transcription Initiation Complex Proteins; D011994:Recombinant Proteins; D012318:RNA Polymerase I
references.citation: Mol Cell Biol. 1996 Nov;16(11):6436-43
references.pmid: 8887672
references.citation: Proc Natl Acad Sci U S A. 2000 Apr 25;97(9):4724-9
references.pmid: 10758157
references.citation: J Gen Virol. 2005 Aug;86(Pt 8):2315-2322
references.pmid: 16033979
references.citation: Genes Dev. 1996 Apr 1;10(7):887-903
references.pmid: 8846924
references.citation: EMBO Rep. 2000 Aug;1(2):171-5
references.pmid: 11265758
references.citation: Cell. 2004 Mar 5;116(5):699-709
references.pmid: 15006352
references.citation: Mol Cell Biol. 1998 Jul;18(7):3752-61
references.pmid: 9632758
references.citation: Gene. 2012 Feb 10;493(2):176-84
references.pmid: 21893173
references.citation: Mol Cell Biol. 2000 Jul;20(14):5269-75
references.pmid: 10866683
references.citation: Cell Growth Differ. 2000 Jan;11(1):63-70
references.pmid: 10672904
references.citation: J Biol Chem. 1996 Aug 30;271(35):21062-7
references.pmid: 8702872
references.citation: Nucleic Acids Res. 1992 Jan 25;20(2):279-85
references.pmid: 1741253
references.citation: Mol Cell. 2004 Feb 27;13(4):573-85
references.pmid: 14992726
references.citation: Trends Biochem Sci. 1999 Nov;24(11):437-40
references.pmid: 10542411
references.citation: Genes Dev. 1996 Oct 15;10(20):2551-63
references.pmid: 8895657
references.citation: Mol Cell. 2010 Mar 26;37(6):809-20
references.pmid: 20347423
references.citation: Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):13458-62
references.pmid: 9391047
references.citation: J Biol Chem. 1998 Dec 11;273(50):33795-802
references.pmid: 9837969
references.citation: EMBO J. 2000 Oct 16;19(20):5473-82
references.pmid: 11032814
references.citation: Genes Dev. 1994 Oct 1;8(19):2349-62
references.pmid: 7958901
references.citation: Science. 2011 Sep 16;333(6049):1637-40
references.pmid: 21921198
references.citation: Science. 1994 Dec 23;266(5193):1966-72
references.pmid: 7801123
references.citation: Mol Cell. 2004 Dec 22;16(6):943-54
references.pmid: 15610737