Figure 2
From: Properties of the ternary complex formed by yeast eIF4E, p20 and mRNA
Determination of dissociation constants (Kd) for eIF4E/p20 wild type complex versus eIF4E/p20 R55-57L, H60-62L (6×) mutant complex bound to capped or uncapped RNA by MicroScale Thermophoresis (MST) and Electrophoretic Mobility Shift Assay (EMSA). (a) MST: 50 nM RED fluorescence dye-labeled eIF4E/His6x-p20 complex (red) or eIF4E/His6x-p20 R55-57L, H60-62L (6×) mutant complex (blue) with serially diluted m7GpppG-capped 64 nt long RNA (40 nt of SSA1 5′UTR), starting at 1 µM. Error bars indicate s.d. (n = 3). (b) EMSA: 600–0 nM His6x-p20 6× mutant/eIF4E complex with 0.25 µM m7GpppG-capped 64 nt long RNA probe (40 nt of SSA1 5′UTR). SYBR Gold Nucleic Acid Gel Stain. GST serves as a negative control. (c) MST: Serially diluted 96 µM eIF4E/His6x-p20 complex (green) or His6x-p20 6× mutant/eIF4E complex (blue) with 50 nM 3′FAM-labeled uncapped 40 nt long 5′UTR of SSA1 RNA. Error bars indicate s.d. (n = 3). (d) Serial dilution of yeast cells carrying p20 knockout (RH2585 ∆p20) or expressing p20 wild type, R55-57L (3×) or R55-57L, H60-62L (6×) mutants. Plates were incubated at 30° or 37 °C for 3 days. Western Blots of whole cells (1/2 OD600), “input” extracts used for m7GDP-Sepharose pulldown, and eluates are shown. Full-length blots are presented in Supplemental Fig. S3. Blots were stained with polyclonal antibodies against eIF4E or p20 as indicated.
