Suppressor analysis of temperature-sensitive RNA polymerase I mutations in Saccharomyces cerevisiae: suppression of mutations in a zinc-binding motif by transposed mutant genes.
Starting with two temperature-sensitive mutants (rpa190-1 and rpa190-5) of Saccharomyces cerevisiae, both of which are amino acid substitutions in the putative zinc-binding domain of the largest subunit (A190) of RNA polymerase I, we have isolated many independent pseudorevertants carrying extragenic suppressors (SRP) of rpa190 mutations. All the SRP mutations were dominant over the corresponding wild-type genes. They were classified into at least seven different loci by crossing each suppressed mutant with all of the other suppressed mutants and analyzing segregants. SRP mutations representing each of the seven loci were studied for their effects on other known rpa190 mutations. All of the SRP mutations were able to suppress both rpa190-1 and rpa190-5. In addition, one particular suppressor, SRP5, was found to suppress two other rpa190 mutations as well as an rpa190 deletion. Southern blot analysis combined with genetic crosses demonstrated that SRP5 maps to a region on chromosome XV loosely linked to rpa190 and represents a transposed mutant gene in two copies. Analysis of the A190 subunit by using anti-A190 antiserum indicated that the cellular concentration of A190 and hence of RNA polymerase I decreases in rpa190-1 mutants after a shift to 37 degrees C and that in the mutant strain carrying SRP5 this decrease is partially alleviated, presumably because of increased synthesis caused by increased gene dosage. These results suggest that the zinc-binding domain plays an important role in protein-protein interaction essential for the assembly and/or stability of the enzyme, regardless of whether it also participates directly in the interaction of the assembled enzyme with DNA.
McCusker
J H
JH
Department of Biological Chemistry, University of California, Irvine 92717.
Yamagishi
M
M
Kolb
J M
JM
Nomura
M
M
D001665:Binding Sites; D015139:Blotting, Southern; D002874:Chromosome Mapping; D015825:Chromosomes, Fungal; D004251:DNA Transposable Elements; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D005838:Genotype; D009154:Mutation; D012318:RNA Polymerase I / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013489:Suppression, Genetic; D013696:Temperature; D015032:Zinc / Q000378:metabolism
D004251:DNA Transposable Elements; D004271:DNA, Fungal; D012318:RNA Polymerase I; D015032:Zinc