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.

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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 Gto-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.The DNA sequences controlling the expression of eukaryotic genes transcribed by RNA polymerases II and III have been recently defined by methods of reverse genetics (1-6). These methods involve the introduction, in vitro, of deletions, substitutions, or point mutations into cloned DNA and the subsequent assay of their effects on the biological activity of the DNA.As yet, little is known about the DNA sequences constituting the RNA polymerase I promoter. Analyses of the template activity of mutants with deletions in the rRNA genes (rDNA) have demonstrated that a major component of this promoter lies upstream of the transcription initiation site (7-9). For mouse rDNA we have shown that 39 nucleotides preceding the start site contain all the sequence information required for efficient, accurate transcription initiation (7). Deletion of five more bases up to position -34 results in a reduction to 1/10th in template activity, while removal of all but 12 nucleotides from the 5' flanking region causes a loss of transcriptional competence. This finding suggests that control sequences lie within the region -12 to -39. However, interpretation of results obtained from deletion mutants is complicated by the fact that essential regions have been removed, thus bringing other sequences abnormally close. Therefore, it is not possible to distinguish between the effects on promoter activity of (i) the deletion and (ii) bringing upstream sequences nearer to the gene. To overcome this difficulty we have constructed a series of mutants containing deletions, substitutions, or base changes within the control region. Using a cell-free transcription system, which allows accurate transcription of mouse rDNA by RNA polymerase I, we have compared the transcriptional efficiencies of wildtype and mutant DNA templates. Functional analyses of these mutants reveal the importance of a guanine at position -16 for pro...

SUMMARYWe 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.