The role of XPB/Ssl2 dsDNA translocation processivity in transcription-start-site scanning

Abstract

The general transcription factor TFIIH contains three ATP-dependent catalytic activities. TFIIH functions in nucleotide excision repair primarily as a DNA helicase and in Pol II transcription initiation as a dsDNA translocase and protein kinase. During initiation, the XPB/Ssl2 subunit of TFIIH couples ATP hydrolysis to dsDNA translocation facilitating promoter opening and the kinase module phosphorylates the C-terminal domain to facilitate the transition to elongation. These functions are conserved between metazoans and yeast; however, yeast TFIIH also drives transcription start-site scanning in which Pol II scans downstream DNA to locate productive start-sites. The ten-subunit holo-TFIIH from S. cerevisiae has a processive dsDNA translocase activity required for scanning and a structural role in scanning has been ascribed to the three-subunit TFIIH kinase module. Here, we assess the dsDNA translocase activity of ten-subunit holo- and core-TFIIH complexes (i.e. seven subunits, lacking the kinase module) from both S. cerevisiae and H. sapiens. We find that neither holo nor core human TFIIH exhibit processive translocation, consistent with the lack of start-site scanning in humans. Furthermore, in contrast to holo-TFIIH, the S. cerevisiae core-TFIIH also lacks processive translocation and its dsDNA-stimulated ATPase activity was reduced ~5-fold to a level comparable to the human complexes, potentially explaining the reported upstream shift in start-site observed in the absence of the S. cerevisiae kinase module. These results suggest that neither human nor S. cerevisiae core-TFIIH can translocate efficiently, and that the S. cerevisiae kinase module functions as a processivity factor to allow for robust transcription start-site scanning.

Ssl2/TFIIH function in Transcription Start Site Scanning by RNA Polymerase II in Saccharomyces cerevisiae

“…Optical tweezer experiments are consistent with TFIIH within the PIC having median processivity on the order of ~90 basepairs, consistent with the average distance of TSSs downstream of yeast TATA boxes. However, purified holo-TFIIH from yeast has much reduced measured processivity and human TFIIH has essentially none 14 . Given that TFIIH activity is predicted to be altered extensively by cofactor interactions in both transcription and nucleotide excision repair (NER), it will be important to understand TFIIH functions within the PIC in vivo.…”

“…Numerous lines of evidence suggest TSS selection by S. cerevisiae Pol II involves a unidirectional scanning mechanism in which the preinitiation complex (PIC) assembles at an upstream core promoter and interrogates consecutive downstream positions for usable TSSs [9][10][11][12][13] . TFIIH is proposed to drive Pol II scanning through ATP-dependent DNA translocase activity 12,[14][15][16][17] . An opticaltweezer-based single molecule analysis of reconstituted S. cerevisiae PICs indicated that an ATP/dATP-induced activity within the PIC causes shortening of the distance between upstream and downstream DNA 17 .…”