An enhancer element in the far upstream spacer region of rat ribosomal RNA gene.

To determine whether far upstream 5'-flanking sequences control rat rDNA transcription, we constructed plasmids containing several 5' and 3' deletions within the nontranscribed spacer region. In vitro transcription of these plasmids identified three enhancer regions, designated A, B, and C, which can dramatically stimulate transcription from the core promoter. Further analysis of region B showed that the enhancer element lies between -2.183 and -2.357 kilobase pairs upstream of the initiation site. The plasmid containing the 174-base pair enhancer element could stimulate rRNA gene transcription as much as 10-20-fold relative to transcription of the plasmid containing only the core promoter. This enhancer was not another promoter domain and could function irrespective of its orientation or distance from the promoter or when inserted downstream of the initiation site. Computer analysis of known sequences of enhancer regions A and C did not reveal any significant homology between these DNA segments and the 174-base pair enhancer element. Competition assay demonstrated that the enhancer element B forms a stable complex with the transcription factor(s) and that interaction between the enhancer and the factor(s) was essential for the stimulation of rDNA transcription. This is the first report of a mammalian ribosomal rDNA enhancer element that exhibits the characteristics of an RNA polymerase II enhancer.