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

Abstract

An RNA polymerase I control region essential for initiation of pre-RNA 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.