Hybrid electron microscopy-FRET imaging localizes the dynamical C-terminus of Tfg2 in RNA polymerase II-TFIIF with nanometer precision.

TFIIF-a general transcription factor comprising two conserved subunits can associate with RNA polymerase II (RNAPII) tightly to regulate the synthesis of messenger RNA in eukaryotes. Herein, a hybrid method that combines electron microscopy (EM) and Förster resonance energy transfer (FRET) is described and used to localize the C-terminus of the second TFIIF subunit (Tfg2) in the architecture of RNAPII-TFIIF. In the first stage, a poly-histidine tag appended to the Tfg2 C-terminus was labeled with nickel-NTA nanogold and a seven-step single particle EM protocol was devised to obtain the region accessible by the nanogold in 3D, suggesting the Tfg2 C-terminus is proximal to the clamp of RNAPII. Next, the C-termini of the Rpb2 and the Rpb4 subunits of RNAPII, adjacent to the clamp, were selected for placing FRET satellites to enable the nano-positioning (NP) analysis, by which the localization precision was improved such that the Tfg2 C-terminus was found to dwell on the clamp ridge but could move to the clamp top during transcription. Because the tag receptive to the EM or FRET probes can be readily introduced to any protein subunit, this hybrid approach is generally applicable to complement cryo-EM study of many protein complexes to nanometer precision.