The Xenopus ribosomal gene enhancers bind an essential polymerase I transcription factor, xUBF.

Abstract

We purified xUBF on the basis of its ability to specifically bind the enhancer elements of the Xenopus laevis rRNA genes. xUBF also binds to both upstream and downstream regions of the X. laevis ribosomal gene promoter and is essential for polymerase I transcription. Unexpectedly, xUBF binds to both upstream and downstream regions of the human ribosomal gene promoter, producing footprints that are indistinguishable from the footprints produced by hUBF, a previously described polymerase I transcription factor isolated from human cells. Despite extensive sequence divergence of vertebrate polymerase I promoters, these data suggest an evolutionary conservation of the primary DNA-protein interaction.

The RNA polymerase I transcription factor UBF is a sequence-tolerant HMG-box protein that can recognize structured nucleic acids

“…In the natural enhancer, the fifteen nucleotide region that was randomized is 80% GC rich, whereas the selected sequences from the random oligo population averaged 42% G+C. This was surprising given our preconception that UBF has a preference for GC rich DNA, a bias stemming from the sensitivity of UBF to competition by poly (dI-dC)poly (dI-dC), but its relative insensitivity to poly (dA-dT) poly (dA-dT) (12,37). We considered the possibility that a UBF recognition site might be located outside of the randomized region of the probe population tested, or even outside of the footprinted region.…”

“…These include Xenopus and mouse enhancers and ribosomal gene promoter domains from Xenopus, human, rat and mouse (discussed in 12, 37). Nonetheless, UBF from all vertebrate species tested produces essentially identical footprints on various promoter or enhancer probes suggesting that vertebrate UBFs recognize DNA in the same way (7,(12)(13)(14)33). To reconcile these observations it has been suggested that the structure of ribosomal gene promoters may have been conserved in evolution without primary sequence conservation (12).…”

“…UBF-DNA interaction conditions and probes DNase I footprinting and gel mobility shift assays (using 4% polyacrylamide, 25 mM TBE gels) involved the use of 5' endlabelled probes ( -0.2ng probe per reaction) and 5-10 ng highly purified UBF as described previously (12,37).…”

“…Xenopus UBF purification UBF was purified from isolated nuclei of an X. laevis kidney cell line (line Xlk2) by chromatography on DEAE -Sepharose, Biorex 70, and Mono Q as described (12,37). UBF-DNA interaction conditions and probes DNase I footprinting and gel mobility shift assays (using 4% polyacrylamide, 25 mM TBE gels) involved the use of 5' endlabelled probes ( -0.2ng probe per reaction) and 5-10 ng highly purified UBF as described previously (12,37).…”

“…Two vertebrate pol I transcription factors, Upstream Binding Factor (UBF) and the TATA-Binding Protein (TBP) have been cloned and characterized (for review see 1). UBF was originally purified by two independent means, first as a human cell fraction necessary for RNA polymerase I transcription in vitro and as an rRNA gene enhancer binding protein (10)(11)(12). UBF has now been purified and cloned from human, frog, rat and mouse (7,(13)(14)(15)(16)(17)(18)(19)(20).…”