Cooperative binding of the Xenopus RNA polymerase I transcription factor xUBF to repetitive ribosomal gene enhancers.
Upstream binding factor (UBF) is a DNA-binding transcription factor implicated in ribosomal gene promoter and enhancer function in vertebrates. UBF is unusual in that it has multiple DNA-binding domains with homology to high-mobility-group (HMG) nonhistone chromosomal proteins 1 and 2. However, a recognizable DNA consensus sequence for UBF binding is lacking. In this study, we have used gel retardation and DNase I footprinting to examine Xenopus UBF (xUBF) binding to Xenopus laevis ribosomal gene enhancers. We show that UBF has a minimum requirement for about 60 bp of DNA, the size of the short enhancer variant in X. laevis. Stronger UBF binding occurs on the longer enhancer variant (81 bp) and on multiple enhancers linked head to tail. In vivo, Xenopus ribosomal gene enhancers exist in blocks of 10 alternating 60- and 81-bp repeats within the intergenic spacer. In vitro, UBF binds cooperatively to probes with 10 enhancers, with five intermediate complexes observed in titration experiments. This suggests that, on average, one UBF dimer binds every two enhancers. A single UBF dimer can produce a DNase I footprint ranging in size from approximately 30 to about 115 bp on enhancer probes of different lengths. This observation is consistent with the hypothesis that multiple DNA-binding domains or subdomains within UBF bind independently, forming more-stable interactions on longer probes.
Putnam
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Biology Department, Washington University, St. Louis, Missouri 63130.
Pikaard
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CS
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA; D004742:Enhancer Elements, Genetic; D008969:Molecular Sequence Data; D011485:Protein Binding; D012318:RNA Polymerase I / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D012269:Ribosomal Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism*; D029867:Xenopus Proteins; D014982:Xenopus laevis
D012269:Ribosomal Proteins; D014157:Transcription Factors; C062332:UBFT protein, Xenopus; D029867:Xenopus Proteins; D004247:DNA; D012318:RNA Polymerase I