Table 5 Peptides used for binding studies
NameSequence (Length)ResiduesKappK (Kinetic), μMKappEq (Equilibrium), μMKapp (Average), μM
CTD-PGGSGGSYSPTpSPSYS (15)CTD2.58 ± 0.651.65 ± 0.062.12
CTDGGSGGSYSPTSPSYS (15)CTDn.d.n.d.>300a
Swi6-NLS-PGGSGGSRELGpSPLKK (15)156−1647.25 ± 4.6310.08 ± 5.108.67
Swi6-NLSGGSGGSRELGSPLKK (15)156−164n.d.n.d.>100a
Whi5-NLS-PGGSGGTPpSPPpSPPGI (15)56−662.87 ± 0.820.48 ± 0.461.67
Whi5-NLSGGSGGTPSPPSPPGI (15)56−66n.d.n.d.>25
Whi5-NES-PGGSGGFLpSPpSPRLRpSPPT (18)152−1643.25 ± 0.71.13 ± 0.182.19
Whi5-NESGGSGGFLSPSPRLRSPPT (18)152−164n.d.n.d.>1500a
  • Amino acids in bold are derived from corresponding protein (residues numbers are indicated). The CTD sequence is based on the consensus heptapeptide repeat in the CTD of Rbp1. Values for KappK and KappEq for the phosphorylated peptides were determined as described in Figure S2. CTD, carboxy-terminal domain; P, phosphorylated peptide; n.d., not determined; NLS, nuclear localization sequences; NES, nuclear export signal; pS, phosphorylated serine.

  • a For the unphosphorylated peptides, KappK and KappEq could not be determined because binding was only significant at the greatest concentration of Ess1 (30 μM). Instead, Kapp was estimated using a standard binding isotherm, F = E/(E + Kapp), where F is the fractional saturation of binding and is E is the concentration of Ess1. Assuming that unphosphorylated and phosphorylated peptides achieve the same signal for saturating binding of Ess1, F is calculated from the ratio U/Psat, where U is the binding signal of unphosphorylated peptide at 30 μM Ess1, and Psat is the signal for saturated binding of the cognate phosphorylated peptide. The binding equation is then solved for Kapp.