IN-VIVO ANALYSES OF THE INTERNAL CONTROL REGION IN THE 5S RIBOSOMAL-RNA GENE FROM SACCHAROMYCES-CEREVISIAE

The internal control region of the Saccharomyces cerevisiae 5S rRNA ge ne has been characterized in vivo by genomic DNase I footprinting and by mutational analyses using base substitutions, deletions or insertio ns. A high copy shuttle vector was used to efficiently express mutant 5S rRNA genes in vivo and isotope labelling kinetics were used to dist inguish impeded gene expression from nascent RNA degradation. In contr ast to mutational studies in reconstituted systems, the analyses descr ibe promoter elements which closely resemble the three distinct sequen ce elements that have been observed in Xenopus laevis 5S rRNA. The res ults indicate a more highly conserved structure than previously report ed with reconstituted systems and suggest that the saturated condition s which are used in reconstitution studies mask sequence dependence wh ich may be physiologically significant. Footprint analyses support the extended region of protein interaction which has recently been observ ed in some reconstituted systems, but mutational analyses indicate tha t these interactions are not sequence specific. Periodicity in the foo tprint provides further detail regarding the in vivo topology of the I nteracting protein.