Skip to main content
. 2010 Aug 30;39(1):59–75. doi: 10.1093/nar/gkq741

Figure 2.

Figure 2.

Ability of the TATA element (TATAAAAA), or a portion of the RPS5 core promoter, to induce ectopic initiation sites within the RPS5 or ADH1 promoters in TAF1 and taf1-N568Δ strains. (A) Schematic diagram showing the insertion sites of the TATA element (upper panel) or a portion (−174 to −81 bp) of the RPS5 promoter (lower panel) within the RPS5 promoter. The number of constructs is shown in parenthesis (upper panel). Note that #60 contains the same promoter fragment (−174 to −81 bp) in duplicate. (B) The ability of each insertion of the TATA element (#48 − #59) or of the −174 to −81 bp fragment (#60) (top) to induce ectopic initiation site(s) in the RPS5 promoter. Primer extension analysis was done as described in Figure 1B, except that the initiation sites of both VTC1 (upper panel) and ADH1 (lower panel, control) were examined. (C) TFIID-dependency of the ectopic initiation sites induced by insertion of the −174 to −81 bp fragment (#60). Total RNA (20 µg) was isolated from the strains carrying a combination of either TAF1 or taf1-N568Δ and either WT or #60 before, or 2 h after, a temperature shift from 30 to 37°C (top). Primer extension analysis was done as described in (B). (D) Transcriptional initiation activities from the sites at −160, −133, −88, −71, −37, −27 and −23 bp (the last three endogenous sites are marked with a horizontal bracket above the ‘WT’) that were examined in (B) and (C) are summarized schematically with vertical black bars in each construct. (E) TFIID-dependency of the ectopic initiation sites induced by insertion of the −174 to −81 bp fragment of the RPS5 promoter at two distinct sites of the ADH1 promoter (−750 to −1 bp). Schematic diagram (upper panel) illustrates the constructs that contain (or do not contain) the −174 to −81 bp fragment of the RPS5 promoter as an insert at the indicated position (−195/−194 or −100/−99 bp) within the ADH1 promoter (#61–63, right) and those that contain an additional TATA to GAGA mutation (#64–66, right). Total RNA (20 µg) was isolated from the strains carrying a combination of either TAF1 or taf1-N568Δ and either WT, #61, #62 or #63 before, or 2 h after, a temperature shift from 30 to 37°C (indicated top, lanes 2–13 and 15–18). Similarly, total RNA (20 µg) was isolated from the strains carrying a combination of TAF1 and either #60, #61, #64, #65 or #66 incubated at 30°C as indicated at the top (lanes 1, 14 and 19–22). Primer extension analysis was done as described in (B).