Structure of the human transcription factor TFIIF revealed by limited proteolysis with trypsin.

In this study, the human general transcription factor IIF (TFIIF), a heteromeric complex of RAP74 and RAP30 subunits, was subjected to limited proteolysis with trypsin. The central region of RAP74 was demonstrated to be highly sensitive to trypsin while both the N- and C-terminal regions contained trypsin-resistant structures. In contrast, RAP30 digestion occurred after proteolysis of RAP74. The digestion pattern of RAP74 recruited into the preinitiation complex showed no marked difference from that of IIF, while RAP30 in the complex was protected from trypsin. These results indicate that RAP74 apparently contains three structural domains, the central one of which is externally surfaced and unstructured, but RAP30 is internally wrapped by RAP74. Furthermore, the accessibility of the central region of RAP74 is unaltered in the minimal preinitiation complex, while RAP30 is involved in promoter recognition through its DNA binding activity.