Figure 1.

Pmh1 is a component of CAK and TFIIH. (A) Extracts from pmh1Δ yeast transformed with pREP41X-FLAG-pmh1⁺ and (B) insect cells expressing FLAG-Pmh1 alone (top) and Flag-Pmh1, Mcs6, and Mcs2 (bottom) were fractionated by Superdex 200 gel-filtration chromatography. Fractions were analyzed by immunoblotting with anti-FLAG antibody. We consistently observed two distinct bands of FLAG-Pmh1 in yeast extracts. (C) Activation of Mcs6 by Pmh1 independent of T-loop phosphorylation. Insect cells were coinfected to express Mcs6-HA (wild type, T160A, S165A, T160A, S165A, and the catalytically inactive K40R/K41R) and His-Mcs2 (present in all lanes). As indicated, cells also expressed Csk1 and/or FLAG-Pmh1. Mcs6 complexes were immunoprecipitated with anti-HA antibody and tested for CTD kinase activity.