Linker scanning of the yeast RNA polymerase I promoter.
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.
Musters
W
W
Biochemisch Laboratorium, Vrije Universiteit, Amsterdam, The Netherlands.
Knol
J
J
Maas
P
P
Dekker
A F
AF
van Heerikhuizen
H
H
Planta
R J
RJ
D001483:Base Sequence; D015152:Blotting, Northern; D002872:Chromosome Deletion; D004275:DNA, Ribosomal / Q000235:genetics*; D005800:Genes, Fungal; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D009154:Mutation; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012318:RNA Polymerase I / Q000235:genetics*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*
D004275:DNA, Ribosomal; D012318:RNA Polymerase I