Promoter contacts by TFB and TFEα during archaeal transcription initiation

Transcription initiation by archaeal RNA polymerase (RNAP) is assisted by transcription factors, TBP, TFB, and TFEα, which show sequence, structural and functional homology to eukaryotic TBP, TFIIB, and TFIIEα. Previous low-resolution cross-linking studies of transcription complexes from the hyperthermophile archaeon Pyrococcus furiosus showed that the transcription bubble DNA makes extensive contacts with TFB and TFEα. Here, contacts between TFB and TFEα surfaces and promoter DNA are mapped with amino acid-to-base precision, using a high-resolution cross-link mapping method. The non-canonical amino acid p-benzoyl phenylalanine (Bpa) was incorporated into TFB1 or TFEα at several positions, transcription complexes were formed and cross-links induced, and the positions of cross-links were mapped by primer extension. In transcription pre-initiation complexes forming a transcription bubble (PICs), the TFB1 B-reader helix and loop conserved sequences interact with the DNA transcribed (T) strand near the upstream side of the transcription bubble, while the B-linker strand conserved sequence interacts with both the transcribed and the non-transcribed (NT) strands. In a 5 nucleotide initially transcribing complex (ITC), cross-links to TFB1 changed for the B-reader but not the B-linker, suggesting scrunching of part of the T strand in the RNAP main channel. The TFEα winged helix cross-linked the NT strand at the upstream edge of the transcription bubble in the PIC. In the ITC, the NT strand changed position dramatically relative to the TFEα wing tip, indicating that the DNA NT strand can bulge out of the main channel as transcription begins and downstream DNA is pulled in. The cross-linking data were compared with structural models of the archaeal PIC and ITC, and indicate that interactions between TFB conserved motifs and DNA are conserved from archaea to humans, while archaeal TFEα contacts to DNA are apparently dissimilar from what is seen in human RNAP II initiation complexes.