A quantitative assay for Xenopus 5S RNA gene transcription in vitro

Abstract

The in vitro transcription of Xenopus 5S RNA genes and of deletion mutants of these genes has been quantitated by assays that measure the efficiency of transcription and the ability to compete with the transcription of a “wild-type” 5S RNA gene. The difference between the competition strength of one repeating unit of X. borealis somatic 5S DNA and its plasmid vector is fifteenfold. tRNA genes and the adenovirus VA RNA genes are weak competitors of 5S RNA synthesis (and vice versa). Deletion of the 5′ flanking region reduces the competition strength of somatic but not oocyte 5S DNA. Except for the influence of the flanking sequence, the regions within the 5S RNA gene that determine competition strength are those that have been shown to interact with a specific transcription factor that is required for accurate initiation of 5S RNA transcription. The major oocyte (Xlo) and trace oocyte (Xlt) 5S RNA genes from X. laevis are transcribed as efficiently as somatic 5S DNAs but compete only one fourth as well. This fourfold difference in the competition strength is due to oocyte-specific base changes within the intragenic control region.