Purification and characterization of a novel factor which stimulates rat ribosomal gene transcription in vitro by interacting with enhancer and core promoter elements.
Previous studies in our laboratory have characterized a 174-base-pair (bp) enhancer sequence in the rat ribosomal DNA spacer region that exhibits all of the characteristics of a polymerase (Pol) II enhancer. Further studies showed that at least half of the enhancer activity resides in a 37-bp motif (E1) within the 174-bp spacer sequence that is located between positions -2.183 and -2.219 kilobase pairs upstream of the initiation site. To identify the factor(s) that binds specifically to the 37-bp enhancer domain, we fractionated whole-cell extract from rat adenocarcinoma ascites cells by chromatography on a series of columns, including an oligodeoxynucleotide affinity column. The final preparation contained two polypeptides of molecular weights 79,400 and 89,100 and was completely devoid of RNA Pol I activity. Electrophoretic mobility shift analysis showed that the polypeptides in the purified preparation (designated E1BF) interacted with both the enhancer element and the core promoter. To determine whether each polypeptide can separately bind to the core promoter and the enhancer, the individual components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, renatured, and subjected to gel retardation analysis. This experiment demonstrated that both polypeptides interacted with the two cis-acting sequences. The specificity of the binding was demonstrated by competition with unlabeled 37-bp and core promoter fragments and lack of competition with nonspecific DNAs in the mobility shift assay. The 37-bp enhancer as well as the downstream sequence of the core promoter were protected by E1BF in the DNase I footprinting assay. Addition of E1BF to limiting amounts of fraction DE-B, which contains all factors essential for Pol I-directed transcription, resulted in three- to fourfold stimulation of ribosomal DNA transcription. Comparison of molecular weights and footprinting profiles did not reveal any relationship between E1BF and other Pol I trans-acting factors.
Department of Pharmacology and Molecular Biology, Chicago Medical School, Illinois 60064-3095.
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Zhang
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Jacob
D000818:Animals; D034961:Antigens, Nuclear; D001483:Base Sequence; D001665:Binding Sites; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000502:physiology; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D066298:In Vitro Techniques; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000302:isolation & purification* / Q000502:physiology; D009841:Oligonucleotides; D011401:Promoter Regions, Genetic; D012335:RNA, Ribosomal / Q000235:genetics*; D051381:Rats; D015183:Restriction Mapping; D014157:Transcription Factors / Q000302:isolation & purification* / Q000502:physiology; D014158:Transcription, Genetic
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009841:Oligonucleotides; D012335:RNA, Ribosomal; D014157:Transcription Factors; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; C000606511:Xrcc6 protein, rat; D000072200:Ku Autoantigen