DNase I footprinting shows three protected regions in the promoter of the rRNA genes of Xenopus laevis.

Abstract

Extracts prepared from Xenopus laevis oocytes contain a protein(s) which specifically protects three discrete regions of the RNA polymerase I promoter from digestion by DNase I. Protected region I, from nucleotide + 15 to nucleotide -10, spans the site of transcription initiation. Protected region II extends from nucleotide -70 to nucleotide -100 relative to initiation, failing within a 42-base-pair sequence which is homologous to the 60/81-base-pair repeated elements which occur outside of the promoter in the spacer. Protected region III is upstream of region II, from nucleotide -120 to nucleotide -140. All three regions correlate with sequences known from deletion studies to be important for promoter function. Deletion mutants which retain either region I or regions II and HI together footprint normally. Deletion of region HI, however, reduces but does not eliminate footprinting on region II, suggesting either that one protein binds to both regions or that the proteins which bind to these sites interact with each other.Transcription of the ribosomal genes of Xenopus laevis is controlled by a promoter that extends from approximately nucleotide -142 to nucleotide +6 relative to the site of transcription initiation (19,28). Sollner-Webb and co-workers (28) have reported that a smaller region from -7 to +6 is able to direct accurate transcription initiation by itself. Although deletion mutants coming in as far as -150 have not been analyzed by electron microscopy, loading of RNA polymerase I at the high density characteristic of ribosomal genes is observed on plasmids truncated to -320 (3). However, two lines of evidence indicate that spacer sequences upstream of -320 can influence expression of these genes. The addition of spacer sequences to ribosomal genes injected into cleaving embryos results in an increased transcription signal from the injected plasmids (5), and when ribosomal genes bearing spacers of different lengths are coinjected into oocytes, the gene bearing the longer spacer is transcriptionally dominant (20,24). We have demonstrated that this effect in oocytes is due to tandemly repeated sequences in the spacer which are called 60/81-base-pair (bp) repeats, and that these spacer sequences have some of the properties associated with enhancers (24). In addition, Labhart and Reeder (14) have demonstrated that a block of 60/81-bp repeats can compete with a promoter when the two are coinjected into oocytes on separate plasmids. This observation suggests that the same protein(s) can bind both to these repeats and to the promoter.The studies described above, in which sequences are manipulated and then assayed in various ways for promoter function, suggest that the promoter of the rRNA genes consists of interacting domains. This view is consistent with models for the promoter structures of the RNA polymerase II and III genes (13, 16). Further, when in vitro transcription extracts are fractionated, activities necessary for correct transcription initiation are, at least in some cases, copurified with a pr...

Two distant and precisely positioned domains promote transcription of Xenopus laevis rRNA genes: analysis with linker-scanning mutants.

“…Our results nicely complement a recent study by Dunaway et al (7) in which the proteins of an X. laevis oocyte extract protect distinct regions of the rDNA promoter in a DNase I footprinting assay. Their protected region III (residues -140 to -120) correlates well with our upstream promoter domain.…”

Footprinting of ribosomal RNA genes by transcription initiation factor and RNA polymerase I.

“…Template commitment also has been demonstrated in mouse and human systems (10)(11)(12). Similarly, crude extracts of Xenopus oocytes contain DNA-binding proteins that alter digestion of rDNA promoter regions (18). However, the inability to demonstrate a correlation between binding and transcriptional activity in the crude system makes interpretation of the results uncertain.…”

Mechanisms of nucleolar dominance in animals and plants.

“…Within each 60/8 l-bp repeat in the spacer is a core element of ~42bp which shares 90% homology with a 42-bp domain within the gene promoter itself (l, 6, 7). The 42-bp domain within the gene promoter is nearly coincident with a proteinbinding domain as defined by DNaseI footprinting (8). In the spacers of a related species, Xenopus borealis, complete 60/ 8 l-bp repeats are absent but several copies of the 42-bp core element are present (9).…”