The RNA polymerase III transcription initiation factor TFIIIB participatesin two steps of promoter opening

Evidence for post-recruitment functions of yeast transcription factor (TF)I IIB in initiation of transcription was first provided by the properties of TFIIIB-RNA polymerase III-promoter complexes assembled with deletion mutant s of its Brf and B " subunits that are transcriptionally inactive because t hey fail to open the promoter. The experiments presented here show that the se defects can be repaired by unpairing short (3 or 5 bp) DNA segments span ning the transcription bubble of the open promoter complex. Analysis of thi s suppression phenomenon indicates that TFIIIB participates in two steps of promoter opening by RNA polymerase III that are comparable to the successi ve steps of promoter opening by bacterial RNA polymerase holoenzyme. B" del etions between amino acids 355 and 421 interfere with the initiating step o f DNA strand separation at the upstream end of the transcription bubble. Re moving an N-terminal domain of Brf interferes with downstream propagation o f the transcription bubble to and beyond the transcriptional start site.