Isolation of thermolabile mutant RNA polymerase II from fission yeast Schizosaccharomyces pombe with mutations in the subunit 3 gene.
Subunit alpha of prokaryotic RNA polymerases plays key roles in protein-protein contacts for both subunit assembly and transcription activation. To gain an insight into the roles of subunit 3, the eukaryotic homologue of alpha, temperature-sensitive mutants of the fission yeast, Schizosaccharomyces pombe, have been isolated after transformation of the mutagenized rpb3 gene encoding mutant subunit 3 of RNA polymerase II. A total of 68 ts mutants were classified into two groups: mutants comprising one group ceased growing immediately after a temperature up-shift, while mutants comprising the other group exhibited delayed growth arrest at high temperatures. RNA polymerase II partially purified from Ts54, one of the group 2 mutants, was thermolabile in vitro, as measured by a non-specific transcription assay. This mutant carries double mutations in domain A of subunit 3, and thus can be used as a reference mutant of RNA polymerase II.
Azuma
Y
Y
Department of Molecular Genetics, National Institute of Genetics, Shizuoka.
Yasui
K
K
Yamagishi
M
M
Ishihama
A
A
D003001:Cloning, Molecular; D004795:Enzyme Stability; D005800:Genes, Fungal; D006358:Hot Temperature; D007700:Kinetics; D009154:Mutation; D012319:RNA Polymerase II / Q000235:genetics* / Q000302:isolation & purification; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D014170:Transformation, Genetic
D012319:RNA Polymerase II