We show that the repetitive 140-base-pair (bp) elements present in the spacer of mouse rRNA genes function as enhancers for RNA polymerase I. Attachment of these elements to the rDNA promoter stimulates rRNA synthesis both in vivo and in vitro. The cis-activating effect of the spacer repeats is orientation-independent and increases with increasing numbers of the 140-bp elements. Competition experiments demonstrate that the spacer repeats bind one or more of the transcription factors interacting with the rDNA promoter. Both the 140-bp elements and the core promoter act cooperatively and thus are functionally linked. The 60/81-bp enhancer repeats from Xenopus laevis rDNA compete for a murine transcription factor(s) and stimulate transcription when fused to the mouse rDNA promoter. The results indicate that despite the marked species specificity of rDNA transcription initiation, common factors may interact with both the rDNA promoter and the enhancer.The intergenic spacers between individual rRNA transcription units have rapidly diverged between different species, varying in length from 2 kilobases (kb) in yeast to >30 kb in mammals. In spite of this marked sequence divergence, several regulatory elements that affect the readout of rRNA genes have been maintained. Functionally important sequence elements within the spacer include promoter and terminator elements, spacer promoters, promoter-proximal terminators, and origins of replication. Furthermore, repetitive blocks of interspersed 60-or 81-base-pair (bp) elements that behave in some respect like enhancer elements have been identified in the intergenic spacer of Xenopus laevis (1, 2). They confer a competitive transcriptional advantage on promoters located in cis but compete against promoters located in trans (3,4). This cis stimulation of transcription is orientation-and position-independent and, therefore, these elements behave like transcriptional enhancers. To date, RNA polymerase I enhancers in rDNA spacers have been rigorously demonstrated only in yeast and in X. laevis and its relative X. borealis. In the mouse spacer there is a cluster of repetitive 140-bp sequence elements (5, 6) that lies between the spacer promoter (7) and the upstream terminator, To (8,9). Both their repetitive nature and their localization between the spacer promoter and the upstream terminator suggest that these elements may be functionally analogous to the frog 60/81-bp element. The results presented here demonstrate that this assumption is correct. We show that the 140-bp repeats stimulate in cis transcription from the rDNA promoter both in a cell-free system and after transfection into mouse cells. This activation appears to be mediated by transcription factor(s) that interact with both the promoter and the enhancer elements. METHODSPlasmid Constructions. See Fig. 1. Plasmid pMrWT contains a 324-bp mouse rDNA promoter fragment (positions -169 to + 155; ref. 10). pMrSP is identical to pMrWT except that the coding region extends to a Pvu II site at +292. The 5'-deletion pl...