Analysis of the rat ribosomal DNA promoter: characterization of linker-scanning mutants and of the binding of UBF.
To investigate the mechanism of transcription of the rat ribosomal DNA (rDNA) promoter, a series of 23 linker-scanning mutants were constructed and assayed in transfected CHO cells and with cell-free extracts. With minor variation, the results of the in vitro and in vivo assays paralleled one another. For example, these assays demonstrated that the mutagenesis of bases from -133 to -124, and those from -106 to -101 of the rDNA promoter significantly inhibited transcription both in vivo and in vitro. Both of these sites lie within the upstream promoter element (UPE) of the rDNA promoter. Several constructs, in particular one that mutated the bases between -49 and -45, were better promoters in vivo than the wild-type promoter. DNAse footprinting experiments with purified UBF, an RNA polymerase I transcription factor, demonstrated the importance of the bases between -106 and -101 for the binding of that factor, providing a positive correlation between the transcription experiments and the binding of UBF to the rDNA promoter.
Xie
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Sigfried and Janet Weis Center for Research, Geisinger Clinic, Danville, PA 17822-2618.
O'Mahony
D J
DJ
Smith
S D
SD
Lowe
D
D
Rothblum
L I
LI
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002457:Cell Extracts; D006224:Cricetinae; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis; D004275:DNA, Ribosomal / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D035463:Pol1 Transcription Initiation Complex Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012318:RNA Polymerase I / Q000378:metabolism; D051381:Rats; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection / Q000235:genetics
D002457:Cell Extracts; D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; D035463:Pol1 Transcription Initiation Complex Proteins; D014157:Transcription Factors; C063844:transcription factor UBF; D004247:DNA; D012318:RNA Polymerase I