Effects of DNA strand breaks on transcription by RNA polymerase III: insights into the role of TFIIIB and the polarity of promoter opening.

Certain deletion mutants of the Brf1 and Bdp1 subunits of transcription factor (TF) IIIB retain the ability to recruit RNA polymerase (pol) III to its promoters, but fail to support promoter opening: deletions within an internal Bdp1 segment interfere with initiation of DNA strand separation, and an N-terminal Brf1 deletion blocks propagation of promoter opening past the transcriptional start site. The ability of DNA strand breaks to restore pol III transcription activity to these defective TFIIIB assemblies has been analyzed using U6 snRNA gene constructs. Breaks in a 21 bp segment spanning the transcriptional start rescue transcription in DNA strand-specific and subunit/mutation-specific patterns. A cluster of Bdp1 internal deletions also reverses the inactivation of transcription with wild-type TFIIIB generated by certain transcribed (template) strand breaks near the transcriptional start site. A structure-based model and topological considerations interpret these observations, explain how Bdp1 and Brf1 help to enforce the general upstream--> downstream polarity of promoter opening and specify requirements for polarity reversal.