Dinucleotide primers facilitate convenient identification of the mouse ribosomal DNA transcription initiation site. A general method for analysis of transcription by RNA polymerases I and III

The in vitro initiation site for RNA polymerase I on the mouse rRNA gene was identified using a new method that is generally applicable to the study of other eukaryotic transcripts. First, the 5' end of mouse rRNA was located to an ApC... by high resolution S1 nuclease mapping. Dinucleotide primers were then used in transcription reactions to demonstrate that this position is the actual de novo initiation site, and not a rapid RNA processing site. For this analysis, initiation was inhibited by reduced rXTP concentration, and, upon supplementation with various dinucleotides, only ApC stimulated correct synthesis. Independently confirming its role as the initiating nucleotide, ATP was shown to be required at a much higher concentration than the other rXTPs for RNA initiation, but not for elongation. These results also demonstrate a marked sequence conservation of rRNA initiation sites between the mouse and frog, two species that violate the general rule of species specificity in RNA polymerase I initiation. Extending these studies to RNA polymerase III, the initiation site for 5 S RNA can be similarly located by dinucleotide analysis and confirmed from the concentration requirements of each rXTP. In addition to allowing initiation at suboptimal rXTP concentration, dinucleotide primers can also circumvent the need for a factor normally required for initiation, suggesting their potential value in dissecting the mechanism of eukaryotic transcription.