Genetic and structural analysis of the essential fission yeast RNA polymerase II CTD phosphatase Fcp1

Lethality of gatekeeper mutations R271A and R299A is rescued by CTD shortening. (A) Exponentially growing cultures of S. pombe strains with the indicated chromosomal fcp1 alleles were adjusted to A₆₀₀ of 0.1 and aliquots of serial fivefold dilutions were spotted to YES agar and incubated at the indicated temperatures. The R271A and R99A alleles were lethal in an rpb1⁺ genetic background. (B) rpb1–natMX cassettes in which the Pol2 CTD was serially truncated from the C terminus to reduce the number of repeats to 26, 20, 18, 16, 13, or 12 were exchanged at the rpb1 locus of heterozygous fcp1⁺, fcp1-R271A–hygMX, and fcp1-R299A–hygMX diploid strains. hyg^R nat^R haploids with the indicated genotypes were recovered after sporulation, revealing that CTD truncation to ≤20 repeats rescued the lethality of R271A and R299A. Serial dilutions of exponentially growing yeast cultures as specified were spotted to YES agar and incubated at the indicated temperatures.