Transcriptional role for the nontranscribed spacer of rat ribosomal DNA.

Abstract

In vitro transcription of the rat rRNA gene led to the identification of a region within a 3.4-kilobase fragment of the nontranscribed spacer (NTS) which significantly increased the transcription of rat ribosomal DNA. Promoter constructs containing this region were transcribed up to 17-fold more efficiently in vitro than templates with only 167 or 286 base pairs of NTS. This effect was also observed when the 3.4-kb fragment of the NTS was subcloned in the opposite orientation and 4 kb upstream of the promoter. The region responsible for the enhanced level of transcription was found between -286 and -1018. The results of order-of-addition experiments suggested that the enhanced level of transcription was the result of the formation of a stable complex between a trans-acting factor and the nontranscribed spacer. DNA-protein binding assays demonstrated that the same region of the NTS determined to have enhancer activity also specifically bound a proteinase K-sensitive factor present in nuclear extracts. The sequence of this region was not found to have any significant homology with the promoter of the rat rRNA gene. This is the first report to assign a transcriptional role to the NTS of a mammalian rRNA gene.

Purification and characterization of a high-mobility-group-like DNA-binding protein that stimulates rRNA synthesis in vitro.

“…Transcription assays were done essentially as described previously (4). To examine the effect of NBP (pl6) on the transcription of rDNA, we preincubated the templates with purified p16 for 5 min before adding extract.…”

“…The spacer or NTS promoter (5,21,40,46), which is much weaker than the 45S (or 40S in Xenopus species) gene promoter, may also play a role in regulating rRNA synthesis. In addition, enhancer elements (4,7,8,27) have been identified at various distances upstream of the transcription initiation sites of the respective genes.…”

“…Studies on the rDNA terminator and enhancer elements suggest that trans-acting factors are involved in these two functions (4,16,44). We have identified the upstream activator sequence (UAS) (or enhancer element) of the rat rRNA gene that is located between nucleotides -1018 (KpnI site) and -286 (BamHI site) (4) and shown that a Hinfl fragment (-1106 to -417) encompassing and overlapping the putative UAS element was recognized by a cellular factor (4). We now report the purification of the DNA-binding protein (referred to as p16) to near homogeneity (>95%) by using a gel electrophoresis DNA-binding assay.…”

“…fourfold, as long as either one or both of the p16-binding sites remained in cis with the promoter. Since one of the p16-binding sites was localized to the region that contained the upstream activator or enhancer sequence (4), as well as the NTS promoter (5), p16 may play a significant role in the regulation of rDNA expression.…”

Species-specific repeat units in the intergenic spacer of the ribosomal RNA cistron of Anopheles aquasalis Curry.

“…27 It has been suggested that these repeat sequences in Drosophila, Daphnia, Rattus, and Xenopus play a role in transcription enhancement of rDNA units. [28][29][30][31][32] The specificity of the repetitive DNA found within the intergenic spacer of the rRNA cistron is perhaps important for the development of specific probes for other species of the subgenus Nyssorhynchus.…”