Electron Microscopy-FRET Imaging Localizes the C-Terminus of the Second TFIIF Subunit in RNA Polymerase II-TFIIF with Nanometer Precision

Abstract

By tightly associating with RNA polymerase II (RNAPII), TFIIF a general transcription factor consisting of Tfg1 and Tfg2 subunit, plays dual roles in both initiation and elongation. Here, we employ a hybrid method of combining nano-probe electron microscopy (EM) and Forster resonance energy transfer (FRET) to investigate the probe-labeled C-terminus of Tfg2 in the RNAPII-TFIIF complex. Single particle EM analysis of nano-gold labeled RNAPII-TFIIF maps the C-terminus of Tfg2 inside the DNA binding cleft or close to the Rpb1 clamp. The FRET distance constraints in-between the pair of Tfg2 and Rpb4, and that of Tfg2 and Rpb2, confines the C-terminus of Tfg2 to the ridge of the Rpb1 clamp. Further FRET measurements followed by nano-positioning analysis reveal that, in the RNAPII-TFIIF elongation complex, the mean position of the C-terminus of Tfg2 shifts to the top of the Rpb1 clamp while, reciprocally, the transcription start site alters its position in the presence of TFIIF. Our results are consistent with the previous studies by crosslinking, establishing that Tfg2 is a malleable protein, and suggesting a dynamical role of Tfg2 in promoter clearance in transcription.