(A) Structural map of Spt6 and Rpb1 mutations that were integrated at their native loci. The abbreviated strain names used in panel B are shown in parentheses. (B) Growth conditions/phenotype (above/below) for the mutations shown in A. Growth on media lacking lysine indicates the Spt⁻ phenotype and growth on media lacking histidine indicates activation of the cryptic promoter within FLO8. Galactose drives elevated transcription of the reporter, so growth on galactose -his indicates a weaker defect than activation on glucose -his. HU150 indicates addition of 150 mM hydroxyurea. Discussion of phenotypes. Mutating the pS1493 interface caused the Spt− phenotype with the severity of the phenotype correlating with the observed effect of the mutation on binding affinity in vitro. The spt6-KK-AA(S), rpb1-S-A double mutant had a stronger phenotype than either single mutant, suggesting that each mutation caused only a partial loss of chromatin maintenance. The additive effects of these alleles suggest that these residues may have additional functions outside of this specific interaction. A more severe Rpb1 mutation that replaces residues LMFSPLV1490-1496 with KMKRRRK1490-1496 (alias = rpb1-FSP+-KRR+) saturated the Spt− phenotype. Because greater phenotypic suppression is not possible, combining rpb1-FSP+-KRR+ with spt6-KK-AA(S) showed no further defect. The rpb1-FSP+-KRR+ strain also activated a cryptic promoter located within the FLO8 gene to a low level (growth on galactose). This defect was enhanced by adding the spt6-KK-AA(S) mutation, indicating that even rpb1-FSP+-KRR+ retains partial Spt6 binding activity or, as mentioned above, that these residues have additional functions that contribute to chromatin maintenance during transcription. Combining the ‘dual site’ spt6 and rpb1 alleles slightly enhanced the phenotypes caused by individual mutations, including activation of the cryptic promoter under low levels of transcription (growth on glucose), suggesting that the individual mutations do not completely disrupt the interaction. This is consistent with the large interface that extends beyond the specific residues mutated in these alleles. We also note the possibility of additional functions of these regions such as interactions of other factors with the Spt6 pockets or Rpb1 residues. These mutants displayed additional defects similar to those caused by a full tSH2 domain deletion, including slow growth at normal and elevated temperatures and hydroxyurea (HU) sensitivity, suggesting that these two interfaces represent essentially all of the biological functions of this domain.