Enhancers for RNA polymerase I in mouse ribosomal DNA.

Abstract

The intergenic spacer of the mouse ribosomal genes contains repetitive 140-base-pair (bp) elements which we show are enhancers for RNA polymerase I transcription analogous to the 60/81-bp repetitive enhancers (enhancers containing a 60-bp and an 81-bp element) previously characterized from Xenopus laevis. In rodent cell transfection assays, the 140-bp repeats stimulated an adjacent mouse polymerase I promoter when located in cis and competed with it when located in trans. Remarkably, in frog oocyte injection assays, the 140-bp repeats enhanced a frog ribosomal gene promoter as strongly as did the homologous 60/81-bp repeats. Mouse 140-bp repeats also competed against frog promoters in trans. The 140-bp repeats bound UBF, a DNA-binding protein we have purified from mouse extracts that is the mouse homolog of polymerase I transcription factors previously isolated from frogs and humans. The DNA-binding properties of UBF are conserved from the mouse to the frog. The same regulatory elements (terminators, gene and spacer promoters, and enhancers) have now been identified in both a mammalian and an amphibian spacer, and they are found in the same relative order. Therefore, this arrangement of elements probably is consequences.widespread in nature and has important functionalThe genes coding for the large rRNAs of most eucaryotes are organized in a similar fashion. From yeast cells to humans, these genes are arranged in multiple tandem copies with precursor-coding regions separated from each other by intergenic spacers (reviewed in references 42 and 54). Recent work from a number of laboratories has suggested that, at least among the multicellular eucaryotes, there is also a broadly conserved arrangement of transcriptional regulatory elements in the spacer (reviewed in reference 49a). The ribosomal genes of the frog, Xenopus laevis, may be considered a paradigm for this type of organization since all of the known regulatory elements have been identified in this organism.A typical intergenic spacer from an X. laevis ribosomal gene is shown in the top line of Fig. 1, with the spacer from a mouse ribosomal gene shown below for comparison. In X. laevis, the intergenic spacer is bounded on the left by a site for 3'-end formation of the precursor (31) and on the right by the gene promoter that directs initiation of the precursor transcript. Between these points are located one or more spacer promoters (4, 41, 53) (the only other known promoters that are recognized by polymerase I), and downstream of the spacer promoters are repetitive 60-and 81-base-pair (bp) elements (60/81-bp elements) that act as enhancers for polymerase I transcription and are additive in effect (11,30,44,48). Between the enhancers and the gene promoter is a termination site (31, 40). The 60/81-bp enhancers bind a Xenopus transcription factor, xUBF, which also binds to the gene promoter (47) and is the frog homolog of human UBF (hUBF; 2) and rat UBF (rUBF; 48a), factors which also stimulate transcription from the gene promoters of these species. The frog...

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

“…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).…”

“…Any model that can accommodate a lack of promoter sequence conservation among species that maintain essentially identical transcription factors must invoke the binding of at least one protein with a relaxed specificity for primary sequence. In this regard, UBF is a likely candidate because we have shown that it is clearly a sequence-tolerant protein previously shown to produce discrete footprints on the promoters of all vertebrate species examined (7,(12)(13)(14)33). In addition, UBF can functionally substitute across species boundaries in some cases (13,14), and it is apparently involved in recruiting or stabilizing the TBP-containing activity in transcription complexes on the promoter (10,13,18,33,34,53).…”

“…Furthermore, mouse enhancers or analogous ribosomal gene sequences from the plant Arabidopsis thaliana can stimulate transcription from a Xenopus promoter in injected frog oocytes (7,8). Likewise, Xenopus enhancers can substitute for their mouse counterparts in vitro (9).…”

“…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). The UBF protein consists of an amino terminal dimerization domain (19, 21,22) followed by five repeated 80 amino acid domains (17) termed HMG-boxes due to their similarity to the DNA binding domains of High Mobility Group (HMG) proteins 1 and 2 (16), and a highly acidic carboxyl terminal tail.…”

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.

“…The best characterized thus far is upstream binding factor (UBF). First purified from a human cell fraction required for RNA pol I transcription in vitro (4), UBF was independently purified from Xenopus laevis as an rRNA gene enhancer-binding protein (11,42) and has since been purified and cloned from human, frog, rat, and mouse cells (1,3,17,19,31,34,43,45,57). Immunological evidence suggests that a UBF homolog is also present in protozoa and plants (51).…”