The RNA polymerase III-recruiting factor TFIIIB induces a DNA bend betweenthe TATA box and the transcriptional start site

TFIIIB, the RNA polymerase III-recruiting factor of Saccharomyces cerevisia e, may be assembled upstream of the transcriptional start site, either thro ugh the interaction of its constituent TATA-binding protein (TBP) with a st rong TATA-box, or by means of the multisubunit assembly factor, TFIIIC. Mis sing nucleoside interference analysis of TFIIIC-dependent TFIIIB-DNA comple x formation revealed enhanced complex formation at 0 degrees C when the DNA is missing nucleosides in two broad 7-10 bp regions centered around base-p airs -17 and -3 relative to the transcriptional start site; no effect of mi ssing nucleosides was evident at 20 degrees C. The implication of these res ults for required DNA flexure in TFIIIC-mediated TFIIIB-DNA complex formati on was pursued in a TFIIIC-independent context, using DNA with a suboptimal 6 bp TATA box (TATAAA). A unique missing nucleoside at the downstream end of the TATA box, corresponding to the position of one of two TBP-mediated D NA kinks, significantly enhances TBP-DNA complex formation. In contrast, TF IIIB displays a broad preference for missing nucleosides within an similar to 15 bp region immediately downstream of the TATA box. Consecutive mismatc hes (4-nt loops), either at the sites of TBP-mediated DNA kinking at both e nds of the TATA box or within the identified region where missing nucleosid es promote TFIIIB-DNA complex formation, also result in enhanced and specif ic TFIIIB assembly; 4-nt loops further downstream do not lead to preferenti al placement of TFIIIB. We conclude that TFIIIB induces an additional DNA d eformation between the TATA box and the start site of transcription that is likely to be more extended than the sharp kinks generated by TBP. (C) 1999 Academic Press.