Functional interactions of the Transcription Factor B during transcription initiation in Pyrococcus furiosus

The preinitiation complex of the transcription machinery in archaeal organisms resembles a simplified version of the eukaryotic RNA polymerase II transcription system. Both systems share homologous general transcription factors to recruit RNA polymerase to the promoter to initiate RNA synthesis. The transcription factor (II)B plays an important role during transcription initiation. Based on eukaryotic cryo-EM and crystal structures several functional interactions and structural transitions of TF(II)B were proposed. To detect specific interactions of the archaeal P. furiosus TFB during transcription initiation different in vitro transcription assays were performed. In addition, the replication protein A of P. furiosus was also investigated using various in vitro experiments. Crosslinking experiments using TFB, which contained a UV inducible photo crosslinker, and site-specific radioactively labeled DNA templates revealed an almost similar topology of the archaeal TFB B-reader and B-linker domains in the preinitiation complex in comparison to corresponding regions predicted in eukaryotic structures. Unlike it was postulated in open complex models, the non-transcribed strand is located closer to the B-linker strand than the B-linker helix. The B-core amino acid F192 contact DNA 19 nucleotides upstream the transcribed strand, in accordance to a published crystal of P. woesei TATA/TBP/TFB-core structure, but is different to predicted eukaryotic closed and open complex models. Crosslinking experiments in stalled complexes showed that RNA interacts with the B-reader loop at a length of 6nt, and further clashes with the B-reader helix domain with a length of 8nt. At register +10 the TFB B-reader is displaced, which causes collapse of the transcription bubble. It was also demonstrated that TFB is present at register +6 to +14 in the complex, and tended to be released from register +15 onwards, indicating a destabilization of TFB at register +13/+14. Alanine substitutions of amino acids of the TFB B-reader loop revealed that this region mainly stabilizes the transcription bubble due to charge-dependent interactions with the transcribing strand. In contrast to the predicted RNA-DNA separation model derived from a eukaryotic initially transcribing complex, RNA-strand separation does not depend on the charge of the PfuTFB B-reader loop. Single molecule FRET experiments revealed that DNA bending depends on the presence of TFB in P. furiosus. In vitro transcription assays with RPA showed that this protein has binding preference to single stranded DNA. Experiments further showed that RPA is not involved in transcription initiation, but it stimulates transcription. Therefore RPA functions during elongation of transcription, possibly due to a stabilization of the RNA polymerase and increase of the processivity. The results presented here give a more detailed insight into molecular interactions of TFB and are the first biochemical data on dynamic rearrangements of TFB during transcription initiation and transition to early elongation. It further deepens the understanding of archaeal transcription processes and complements structural information derived from related eukaryotic organisms

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