The Saccharomyces cerevisiae RNA polymerase III recruitment factor subunits Brf1 and Bdp1 impose a strict sequence preference for the downstream half of the TATA box

Abstract

Association of the TATA-binding protein (TBP) with its cognate site within eukaryotic promoters is key to accurate and efficient transcriptional initiation. To achieve recruitment of Saccharomyces cerevisiae RNA polymerase III, TBP is associated with two additional factors, Brf1 and Bdp1, to form the initiation factor TFIIIB. Previous data have suggested that the structure or dynamics of the TBP-DNA complex may be altered upon entry of Brf1 and Bdp1 into the complex. We show here, using the altered specificity TBP mutant TBPm3 and an iterative in vitro selection assay, that entry of Brf1 and Bdp1 into the complex imposes a strict sequence preference for the downstream half of the TATA box. Notably, the selected sequence (TGTAAATA) is a perfect match to the TATA box of the RNA polymerase III-transcribed U6 small nuclear RNA (SNR6) gene. We suggest that the selected T*A base pair step at the downstream end of the 8 bp TBP site may provide a DNA flexure that promotes TFIIIB-DNA complex formation.

Figure 1

Determination of the dissociation rate constant, k_off. (A) TATA DNA was incubated with TBPm3 for 1 h, and aliquots loaded on the gel at time t after addition of poly(dA–dT):poly(dA–dT). C indicates the TBPm3–DNA complex and F indicates the free dsDNA. The faint band below F is ssDNA. (B) Rate of complex dissociation. The fraction of complex corrected for dissociation during electrophoresis is shown as a function of time after addition of competitor.

Figure 2

Determination of the association rate constant, k_on. (A) TBPm3 (50 nM) and TGTA DNA were incubated for time t (10 s to 10 min) and loaded on the gel immediately after addition of competitor to determine the apparent first-order rate constant, k_obs. C indicates the TBPm3–DNA complex and F indicates the free dsDNA. (B) The kinetics of TBPm3–DNA complex formation were determined at the indicated protein concentrations. The reciprocal of k_obs is plotted as a function of the reciprocal of the TBPm3 concentration.

Figure 3

Alignment of sequences selected by TBPm3. Only sequences retaining the original TGTAA sequence are shown. Bases corresponding to randomized positions are in boldface.

Figure 4

TBPm3 binds to DNA representing the selected sequence. Of each 26 bp 50 fmol DNA was incubated with 0, 100, 200, 500 and 1000 fmol TBPm3. Left panel, selected sequence, right panel, TGTA probe used for affinity determinations. C indicates the TBPm3–DNA complex and F indicates the free dsDNA.

Figure 5

MPE-Fe(II) 2D footprinting confirms binding of TBPm3 at the TGTA box. Densitometry profiles of 76 bp DNA containing the favored sequence (TGTAAATTG) incubated with (black line) and without (blue line) TBPm3 show protection at the TGTA box. Numbering is based on the start site of transcription (+1). Gray line represents uncut DNA.

Figure 6

Alignment of sequences selected by TFIIIB. Only sequences retaining the original TGTAA sequence are shown. Bases corresponding to randomized positions are in boldface.