Structures of Transcription Complexes
Transcription initiation in bacteria requires RNA polymerase (RNAP) and the transcription initiation factor sigma. The bacterial transcription initiation complex contains six polypeptides (five in RNAP, one in sigma) and promoter DNA, and has a molecular mass of 0.5 MDa.
Understanding bacterial transcription initiation requires understanding the structures of polypeptides in bacterial transcription initiation complexes and the arrangements of these polypeptides relative to each other and relative to promoter DNA.
We are using cryogenic electron microscopy (cryo-EM) and x-ray crystallography to determine atomic structures of transcription initiation complexes, ensemble and single-molecule fluorescence resonance energy transfer (FRET) to define distances between pairs of site-specifically incorporated fluorescent probes, photocrosslinking to define polypeptides near site-specifically incorporated photocrosslinking probes, and protein footprinting and residue scanning to define residues involved in contacts. In support of these activities, we are developing procedures to incorporate fluorescent probes and photocrosslinkers at specific sites within large multisubunit nucleoprotein complexes, and we are developing automated docking algorithms to integrate structural, biophysical, biochemical, and genetic data in order to construct models for structures of complexes.