Displacement of the transcription factor B reader domain during transcription initiation

Transcription initiation by archaeal RNA polymerase (RNAP) and eukaryotic RNAP II requires the general transcription factor (TF) B/ IIB. Structural analyses of eukaryotic transcription initiation complexes locate the B-reader domain of TFIIB in close proximity to the active site of RNAP II. Here, we present the first crosslinking mapping data that describe the dynamic transitions of an archaeal TFB to provide evidence for structural rearrangements within the transcription complex during transition from initiation to early elongation phase of transcription. Using a highly specific UV-inducible crosslinking system based on the unnatural amino acid para-benzoyl-phenylalanine allowed us to analyze contacts of the Pyrococcus furiosus TFB B-reader domain with site-specific radiolabeled DNA templates in preinitiation and initially transcribing complexes. Crosslink reactions at different initiation steps demonstrate interactions of TFB with DNA at registers +6 to +14, and reduced contacts at +15, with structural transitions of the B-reader domain detected at register +10. Our data suggest that the B-reader domain of TFB interacts with nascent RNA at register +6 and +8 and it is displaced from the transcribed-strand during the transition from +9 to +10, followed by the collapse of the transcription bubble and release of TFB from register +15 onwards.

Transcription is a cyclic process in which information derived from DNA is transcribed into RNA molecules by RNA polymerases (RNAPs). Cellular RNAPs need basal transcription factors to carry out promoter-dependent RNA synthesis. The minimal preinitiation complex (PIC) of the eukaryotic RNAP II system consists of six general transcription factors (TFIID, TFIIA, TFIIB, TFIIE, TFIIF and TFIIH) representing a complex and highly regulated transcription machinery. The archaeal transcription system constitutes a simplified version of that of eukaryotes. Its preinitiation complex consists of RNAP, the TATA-box binding protein (TBP), transcription factor B (TFB) and E (TFE), which are structurally and functionally related to eukaryotic RNAP II, TBP, TFIIB and TFIIEα.