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

Abstract

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...

Structural determinant of the species-specific transcription of the mouse rRNA gene promoter.

“…This is supported by the footprints as well as competition data described above. The conserved guanine residue at nucleotide -16 has an important role in this binding, as shown by previous studies (15,30,33; Tanaka et al, unpublished observation). On the other hand, conserved nucleotides -1 and -7 are presumably crucial for the relatively speciesindependent recognition processes of rDNA such as the binding and functioning of polymerase I enzyme or other factor(s) (3,14,30).…”

Two distinct promoter elements in the human rRNA gene identified by linker scanning mutagenesis.

“…The transcription phenotypes of the spacing and inversion mutations indicate that the role of the upstream control element depends on the core element in a manner which is both directional and distance dependent. The importance of the upstream element for transcription was first observed in in vitro reactions with deletion mutants, but recently it has also been demonstrated by an in vivo transfection assay (29 (4,10,28,33). Recently, the mouse upstream element has been mapped in greater detail to a position similar to that of the human upstream element (21).…”

A 140-base-pair repetitive sequence element in the mouse rRNA gene spacer enhances transcription by RNA polymerase I in a cell-free system.

“…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.…”