Cooperative Binding of the Xenopus RNA Polymerase I Transcription Factor xUBF to Repetitive Ribosomal Gene Enhancers

Abstract

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.

The RNA Polymerase I Transactivator Upstream Binding Factor Requires Its Dimerization Domain and High-Mobility-Group (HMG) Box 1 To Bend, Wrap, and Positively Supercoil Enhancer DNA

“…recognize structural features of nucleic acids rather than specific sequences. Despite UBF's promiscuous DNA-binding behavior, UBF binding to rRNA gene enhancer sequences is thought to be biologically significant (41)(42)(43)47). Supporting evidence is that enhancers will compete in trans against an rRNA gene promoter both in vitro and in injected oocytes (27,42), and addition of highly purified UBF can overcome this enhancer competition phenomenon in vitro (42).…”

“…Supporting evidence is that enhancers will compete in trans against an rRNA gene promoter both in vitro and in injected oocytes (27,42), and addition of highly purified UBF can overcome this enhancer competition phenomenon in vitro (42). Therefore, we have used enhancer probes to study UBF-DNA interactions in some detail (10,42,47). UBF binds the X laevis enhancers (and other probes of equal length) with a stoichiometry of one dimer for every two enhancers (-140 bp).…”

“…UBF binds the X laevis enhancers (and other probes of equal length) with a stoichiometry of one dimer for every two enhancers (-140 bp). On probes consisting of four or more enhancers, UBF dimers bind cooperatively presumably as a result of protein-protein interactions, conformational changes in the target DNA that affect the free energy of UBF binding, or both (47). Single UBF dimers bind probes varying in length from -60 to 150 bp but fail to bind DNA fragments shorter than about 60 bp (47).…”

“…On probes consisting of four or more enhancers, UBF dimers bind cooperatively presumably as a result of protein-protein interactions, conformational changes in the target DNA that affect the free energy of UBF binding, or both (47). Single UBF dimers bind probes varying in length from -60 to 150 bp but fail to bind DNA fragments shorter than about 60 bp (47). However, within the 60 to 150-bp size range, the strength of UBF binding is proportional to probe length (47).…”

“…Single UBF dimers bind probes varying in length from -60 to 150 bp but fail to bind DNA fragments shorter than about 60 bp (47). However, within the 60 to 150-bp size range, the strength of UBF binding is proportional to probe length (47).…”