Linker scanning of the yeast RNA polymerase I promoter

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Abstract
To define the RNA polymerase I promoter in the rDNA of Saccharomyces cerevisiae more precisely, we have constructed a series of 5′- and 3′-deletion mutants in a novel, plasmid-borne rDNA minigene, that also contains the transcriptional enhancer. Our data show that the Pol I promoter, in this context, extends from position −155 to +27, with 5′-deletions up to −134 and 3′-deletions up to −2 removing essential sequence information. To investigate the internal organization of the yeast Pol I promoter, linker scanning mutants were constructed, that traverse the Pol I promoter region and comprise between 5 and 12 clustered point mutations. Analysis of minigene transcription in yeast cells transformed with these plasmids demonstrates that the Pol I promoter consists of three domains. Mutations in Domain I (from position −28 to +8) and Domain II (−70 to −51) drastically reduce promoter activity, whereas clustered point mutations in Domain III (starts at position −146 and presumably extends to position −76) appear to have less effect. Furthermore, the insertion of 4 nt between Domains I and II diminishes minigene transcription, indicating that the relative positions of these domains is essential.