Additional RNA polymerase I initiation site within the nontranscribed spacer region of the rat rRNA gene.

Abstract

We identified and characterized an additional promoter within the nontranscribed spacer (NTS) of the rat ribosomal gene repeat that is capable of supporting initiation of transcription by RNA polymerase I in vitro. Within this promoter there is a sequence of 13 nucleotides which is 100% homologous to nucleotides -18 to -6 (+1 being the frst nucleotide of 45S rRNA) of the major promoter of 45S pre-rRNA and is located between nucleotides -731 and -719. To identify the exact location of the upstream initiation site, the RNA synthesized in vitro from this new promoter was gel isolated and subjected to fingerprint analysis, Southern hybridization, and reverse transcriptase elongation. Based on these analyses, the in vitro-synthesized RNA initiates with an A at nucleotide -713. When compared individually, the upstream promoter was transcribed ninefold less efficiently than the major promoter. When templates which contain both promoters on the same piece of DNA were transcribed, the major promoter was at least 50-fold more efficient.Eucaryotic ribosomal genes are arranged as tandemly repeating units. The transcription units that code for the RNA precursor are flanked by the nontranscribed spacer (NTS) regions. The transcribed portion of the repeat consists of an external transcribed spacer, the region that codes for 18S rRNA, an internal transcribed spacer, the 5.8S coding region, a second internal transcribed spacer, the region that codes for 28S rRNA, and a short 3' external transcribed spacer (23).Besides functioning to direct and regulate transcription, the NTS has been both proposed and shown to have roles in recombination (39), DNA replication (37), and chromatin structure (8).By far the greatest number of studies on the NTS have focused on its role in transcription. The DNA sequences immediately upstream of the rRNA transcription initiation site have been intensively studied by several laboratories. A core promoter region, located between nucleotides -39 and +6, has been identified, and specific nucleotides within this region have been shown to be required for initiation (11, 18, 2,, 25, 38, 43, 47). A second region, the upstream control element or upstream promoter element (between nucleotides 150 and -110), has been demonstrated to stabilize effectively the preinitiation complex (15,19,30,44,45; B. G. Cassidy, R. Haglund, and L. I. Rothblum, Biochim. Biophys. Acta, in press).In yeasts and Xenopus laevis, regions several hundred to several thousand nucleotides upstream of the 5' end of the ribosomal precursor have been shown to have profound effects on promoter utilization and the efficiency of transcription. In yeasts, a polymerase I promoter (42) or enhancer element (9) has an effect on the transcription of the 35S rRNA precursor (10). Two promoter-related elements present in multiple copies in the NTS of X. laevis have also been identified (reviewed in references 7 and 34). These elements, the Bam islands and the 60-or 81-base-pair (60/81-bp) repeats, affect the utilization of the polymerase I promoter and ...

c-Myc induces changes in higher order rDNA structure on stimulation of quiescent cells

“…c-Myc induces rDNA gene loops C-N Shiue et al associated factors to sequences upstream of the main promoter probably reflects the presence of a secondary promoter in this upstream region (Cassidy et al, 1987). The binding of TBP to the R þ 1 region is consistent with an earlier study showing binding of SL-1 within the transcribed region (Mais et al, 2005), but it should also be noted that other studies have not identified TBP binding within the transcribed region (Grandori et al, 2005).…”

Analysis of the rat ribosomal DNA promoter: characterization of linker-scanning mutants and of the binding of UBF

“…It has been shown that the phosphorylation of UBF is dependent upon the presence of growth factors in the media (25), and more than one domain of UBF is phosphorylated (39). It was also found that treatment of purified UBF with alkaline phosphatase reduced its ability to activate transcription in vitro (25 (5,38). Furthermore, analysis of the effects of deletion mutants and linker-scanning mutants of the upstream promoter elements of the rat and human promoters demonstrated that an intact UPE was required for the maximal effect of UBF in vitro (5,17,26).…”

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

“…200 base pairs (bp) upstream of the initiation site (referred to as To in the mouse or as T3 in the Xenopus rDNA repeat) may act as an upstream element of the adjacent promoter (15,22,36). 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.…”

“…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.…”