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Nucleic Acids Research, 1989, Vol. 17, No. 23 9661-9678
© 1989

MOLECULAR BIOLOGY

Linker scanning of the yeast RNA polymerase I promoter

W. Musters, J. Knol, P. Mass, A.F. Dekker, H. van Heerikhuizen and R.J. Planta*

Biochemisch Laboratorium, Vrije Universiteit de Boelelaan 1083, 1081 MV Amsterdam, The Netherlands

*To whom correspondence should be addressed

Received September 1, 1989. Revised October 31, 1989. Accepted October 31, 1989.

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.


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