The core promoter of mouse rDNA consists of two functionally distinct domains.

We have determined the sequences constituting the minimal promoter of mouse rDNA. A very small region immediately upstream of the transcription start site (from -1 to -39) is sufficient to direct correct transcription initiation. Sequences immediately downstream of the transcription start site (+1 to +11) increase the efficiency of transcription initiation. Point mutations within the core promoter have been generated and assayed for their effects on template activity and on interaction with the pol I specific transcription factor TIF-IB. The core promoter element appears to consist of two functionally different domains. The distal sequence motif from position -22 to -16 is recognized by factor TIF-IB. Mutations within this region lead to similar changes of both template activity and binding of TIF-IB. Two point mutations within the proximal sequence motif from -15 to -1 do not affect TIF-IB binding although they severely impair transcription initiation. It is suggested, that this proximal region plays a role in the assembly of functional transcription initiation complexes rather than in the primary binding of TIF-IB.

A repeated 18 bp sequence motif in the mouse rDNA spacer mediates binding of a nuclear factor and transcription termination.

DNA sequences and protein factors directing termination of mouse rDNA transcription in a nuclear extract system were examined. Termination is specific and requires a sequence element AGGTCGACCAGATTANTCCG (the Sall box) that is present eight times in the spacer region downstream of the 3' end of pre-rRNA. Exonuclease III protection experiments reveal the binding of a nuclear protein to the Sall box. Deletions, insertions, and point mutations in the Sall box reduce or abolish the interaction with the nuclear factor and disrupt transcription termination. A synthetic oligonucleotide corresponding to the Sall box consensus sequence governs transcription termination in vitro, although with reduced activity. Therefore, other sequences normally surrounding the Sall box appear to contribute to the accuracy and efficiency of termination.

Transcription of mouse rDNA terminates downstream of the 3' end of 28S RNA and involves interaction of factors with repeated sequences in the 3' spacer.

RNA polymerase I terminates transcription of mouse rDNA 565 bp downstream of the 3' end of mature 28S rRNA. This specific termination event can be duplicated in a nuclear extract system. RNA molecules with authentic 3' ends are transcribed from ribosomal minigene constructs provided the templates retain a minimal length of downstream spacer sequences. The nucleotide sequence of the region of transcription termination contains a set of repetitive structural elements consisting of 18 bp conserved nucleotides surrounded by stretches of pyrimidines. Termination in vivo occurs within the first element. This site is preferentially used in vitro at low template concentrations. At increasing DNA concentrations a termination site within the second repetitive element is used. Competition experiments with defined 3'-terminal fragments suggest that transcription termination by RNA polymerase I requires interaction of some factor (or factors) with the repetitive structural elements in the 3' nontranscribed spacer.

In vitro mutagenesis and transcriptional analysis of a mouse ribosomal promoter element.

An RNA polymerase I control region essential for initiation of pre-rRNA transcription has been identified by mutagenesis in vitro of mouse rDNA (ribosomal RNA genes) and transcription in a cell-free system derived from Ehrlich ascites cells. Substitution of nucleotides between -35 and -14 by foreign DNA sequences caused a loss of template activity, which indicates that an important promoter element is located within this region. To identify the nucleotides essential for RNA polymerase I function, single and multiple point mutations within this control region were generated and the modified DNAs were assayed for template activity. The phenotypes of mutants in which C-to-T transitions have been introduced at positions -36, -31, -27, -22, -21, and -13 were identical to the wild type. Conversion of G to A at position -15 resulted in a 20% increase of promoter activity, whereas a G-to-A transition at -16 decreased transcription by 95%. Competition experiments between mutant and wild-type DNAs suggest that the guanine at -16, which is evolutionarily highly conserved, interacts with essential components of the transcription apparatus.