49. Amino Acid Substitution of the Largest Subunit of Yeast RNA Polymerase II: Effect of a Temperature-sensitive Mutation Related to G1 Cell Cycle Arrest
Kimihiko Sugaya
Keywords: RPB1, site-directed mutagenesis, temperature sensitivity, distribution
A temperature-sensitive mutant, tsAF8, of the BHK21 Syrian hamster cell line shows cell cycle arrest at nonpermissive temperatures in mid-G1 phase. DNA sequence comparison of the largest subunit of RNA polymerase II (Rpb1) from the wild type and the mutant shows that the mutant phenotype results from a (hemizygous) C-to-A variation at nucleotide 944 in one rpb1 allele; this gives rise to an Ala-to-Asp substitution at residue 315 in the protein. To examine this hemizygous mutation in the rpb1 gene and to determine the molecular mechanism underlying the temperature-sensitive defects, this amino acid substitution was introduced into the Schizosaccharomyces pombe rpb1 gene. Whereas tsAF8 cells showed growth defects and an altered distribution of Rpb1 at nonpermissive temperatures, yeast cells harboring this amino acid substitution did not show any temperature sensitivity. Distribution of Rpb1 in yeast cells appeared normal at permissive and nonpermissive temperatures. The additive or subtractive effect of another temperature-sensitive Rpb1 mutation was also very little. These results suggest that mutation of the rpb1 gene, which is critical in mammalian cells, may not be deleterious in yeast cells.