Isolation of yeast minimal open complexes and regulation by TFIIF.
Human Pol II open complexes can be formed in vitro
by two methods. In the first method, PICs containing Pol II and all the general transcription factors are incubated with ATP or dATP, leading to the unwinding of ~10 bp surrounding the transcription start site, monitored by KMnO4
reactivity with single-stranded DNA (13
). The maintenance of this state requires the continued hydrolysis of ATP, since the addition of an excess of ATPγS reverts the open complex back to the PIC (8
). In contrast, the ATP addition to S. cerevisiae
PICs has not yet been observed to generate KMnO4
-sensitive DNA between TATA and the transcription start site. One possible reason for this is that ATP may also induce start site scanning so that the single-stranded DNA is not localized to a single position.
An alternative method of open complex formation involves assembling factors on promoter DNA containing a preformed heteroduplex bubble (12
). In the human system, the optimal position for the bubble is variable, depending on the promoter used, but is generally located from positions ~−9 to +2 relative to the transcription start site. Transcription from these complexes requires only the factors TBP, TFIIB, and Pol II (20
). TBP and TFIIB are presumably necessary to tether Pol II near the heteroduplex DNA and to assist in the positioning of the DNA within the Pol II active site. TFIIF was reported previously to either stimulate or have little effect on the activity of these minimal human open complexes (20
). TFIIE and TFIIH are unnecessary for the activity of human heteroduplex complexes, probably because they act primarily in DNA strand separation and/or the stabilization of the open state.
Yeast transcription initiates at variable distances downstream from the site of PIC formation, and it is not clear why the yeast system does not initiate at the same position as that of human Pol II. One model consistent with previous results is that initiation at ~30 bp downstream from TATA is blocked, forcing Pol II to scan downstream sequences for an appropriate start site. Because of this behavior, it was not clear whether yeast Pol II open complexes could be formed by using heteroduplex templates and, if so, where best to position the single-stranded bubble. To test whether open complexes could be formed, a series of 10 heteroduplex templates was generated based on the yeast HIS4
promoter, each containing 12 bases of unpaired single-stranded DNA. These bubbles span the region beginning 18 bp downstream of TATA, through the normal HIS4
initiation sites ~80 bp downstream of TATA (A). The promoter derivatives also contained an additional 372 and 165 bp of upstream and downstream DNA (22
) and were attached to magnetic beads via biotin at the 5′ end of the promoter. The major most upstream HIS4
transcription start is defined as position +1.
We initially characterized the activities of two bubble templates, one coincident with the position of mammalian transcription initiation (bubble 3) and the other overlapping the normal HIS4 transcription start site (bubble 15). B shows the activity of the bubble 3 heteroduplex template compared to transcription using the double-stranded HIS4 promoter. In all experiments, nucleotides were added for 3 min to preformed protein-DNA complexes to limit transcription to approximately one round of initiation. B (lanes 1 and 2) shows that VP16 activated transcription using yeast nuclear extracts on the double-stranded HIS4 template. This transcription activity is comparable to the level of basal transcription (no activator) using a system containing highly purified and recombinant yeast factors (TFs) (B, lanes 3 and 4). As expected, both the crude and reconstituted complete systems require hydrolyzable ATP, as ATPγS, a substrate for RNA synthesis but not open complex formation, does not promote transcription when substituted for ATP (B, lanes 2 and 4).
Very high levels of transcription were observed using both the bubble 3 and bubble 15 promoters (B and C). High-level transcription from bubble 3 required Pol II, TBP, and TFIIB (B, lanes 5 to 8), and a similar behavior was observed with bubble 15. As expected, transcription initiation from these promoters was independent of β-γ-hydrolyzable ATP, since the substitution of ATPγS for ATP gave similar levels of mRNA (C, lanes 1, 2, 7, and 8).
Unexpectedly, transcription from bubble 3 using the complete set of general factors was 3-fold lower than that from the minimal system (C, lanes 1, 2, 5, and 6). This repression appeared to be caused by TFIIF, since the addition of TFIIF to the minimal set of factors also repressed transcription (C, lane 4). In contrast, TFIIF had no effect on transcription from bubble 15 at the normal site of HIS4 initiation (C, lanes 7 to 12). Our results demonstrate a surprising flexibility of the yeast system for the position of the heteroduplex bubble, ranging over ≥60 bp, and show that there is nothing preventing the minimal set of Pol II factors from initiation at the position used by mammalian Pol II. Importantly, TFIIF inhibits initiation by yeast Pol II at the mammalian start site position in heteroduplex HIS4 templates. This mechanism may also contribute to the inhibition of initiation from the mammalian start site position in double-stranded DNA when all factors are present.
To further investigate the flexibility of open complexes and the ability of TFIIF to repress initiation, the complete set of heteroduplex templates was tested for transcription activity and repression by TFIIF (A). Bubbles 1 to 7, spanning the mammalian start site position from 18 to 42 bp downstream of TATA, were all active as templates for the minimal set of factors. Bubble 7 had a significant background of transcription from Pol II alone, while the other templates all gave significantly higher levels of transcription when TBP and TFIIB were added. Transcription from all these templates was repressed by the addition of TFIIF. In contrast, bubbles 9, 10, and 12 (single-stranded DNA 42 to 65 bp from TATA) gave little or no transcription. Finally, bubbles 14 to 16, which all overlap the normal HIS4 initiation sites, gave high levels of initiation that were either stimulated by or indifferent to the addition of TFIIF. Bubble 14 also had a high background level of transcription from Pol II alone.
Fig 2 Open complex activity and response to TFIIF at heteroduplex bubbles spanning the HIS4 promoter. (A) In vitro transcription, visualized using primer extension, from 10 heteroduplex bubbles with Pol II alone or TBP/TFIIB/Pol II (minimal open complexes) (more ...)
An analysis of initiation from bubble 3 using a high-resolution gel showed that initiation in the absence of TFIIF begins from sites within and just downstream of the single-stranded region and that TFIIF has its strongest repressive effect on starts within single-stranded DNA (B, with brackets indicating the region of single-stranded DNA). In contrast, transcription from bubble 15 initiates almost entirely within the single-stranded region, and transcription from at least two initiation sites is stimulated by TFIIF.
To test whether TFIIF repressed transcription initiation from bubble 3 rather than a later step, such as the transition to the elongation mode, minimal open complexes were incubated with ATP, CTP, and [α-32P]UTP for 30 min, generating a series of short RNAs (, lane 2). The synthesis of these short RNAs was inhibited by TFIIF, showing that TFIIF inhibits initiation (, lane 3). In contrast, the addition of TFIIF stimulated the production of short RNAs from bubble 15 and the nucleotides CTP, UTP, and [α-32P]GTP (, lanes 5 and 6). Thus, TFIIF appears to act by the modulation of transcription initiation.
Fig 3 TFIIF represses the transcription initiation activity of the open complex. In vitro transcription from bubbles 3 and 15 by minimal open complexes with or without TFIIF was initiated with limiting nucleotides for 30 min. An α-32P-labeled nucleotide (more ...) Sequence of the heteroduplex bubble determines the response to TFIIF.
We next investigated why transcription from the different bubble templates showed different responses to TFIIF. Possible variables include the distance of the bubbles from TATA or DNA sequence differences upstream of and/or within the bubbles. To test if the sequence upstream of the single-stranded region was important for regulation by TFIIF, 12 bp of DNA upstream of bubble 3 (repressed by TFIIF) was replaced by 12 bp upstream of bubble 15 (stimulated by TFIIF) (bubble 3 positions −54 to −43) (A). The replaced upstream DNA is underlined in A. Transcription from this new promoter variant was still repressed to the same extent by the addition of TFIIF compared to bubble 3 (B, lanes 1 to 4), showing that the sequence upstream of the bubble has no effect on the response to TFIIF. To test the importance of the bubble sequence for the TFIIF response, we replaced the single-stranded bubble 3 sequence with that of bubble 15 (bubble 3::15) (A). Surprisingly, we found that transcription from this promoter variant was slightly stimulated by TFIIF (B, lanes 7 and 8). High-resolution gel analysis showed that initiation from this template used two primary start sites, at positions −34 and −32, and that the TFIIF addition caused a strong preference for the transcription start site at position −32 (C, lanes 5 and 6).
Fig 4 The sequence of bubble 3 determines the response to TFIIF. (A) Sequences of the HIS4 promoter and bubble 3 variants. Highlighted are the TATA box (in red), the hypothetical human transcription start site position (in green), and S. cerevisiae start sites (more ...)
Since TFIIF altered the sequence preference of Pol II at the transcription start site, we tested the effect of changing the base at one of these TFIIF-dependent starts. At bubble 3, initiation was evenly distributed among three start sites, with two being within the single-stranded bubble (C, lane 1). Upon the addition of TFIIF, transcription within the bubble was repressed, while initiation within double-stranded DNA at −29G was only modestly repressed. However, if −29G was changed to T (a nonpreferred base [bubble G −29T]), the addition of TFIIF repressed nearly all transcription, since there was no optimal transcription start site remaining (B, lanes 5 and 6, and C, lanes 3 and 4). These results show that TFIIF imposes a strong and unexpected sequence preference on the transcription start site.
Both sequence and distance of the bubble from TATA contribute to the efficiency of initiation.
We next investigated why transcription initiates poorly from the bubbles located between the mammalian initiation position and the normal HIS4 transcription start sites (bubbles 9 to 12; 42 to 65 bp downstream from TATA) (A). One possibility is that this region is at a nonoptimal distance from TATA and perhaps generates a strained, inactive open complex. Alternatively, the DNA sequence within this region may not be a good substrate for initiation. Several promoter variants were constructed to test these two possibilities (A). The single-stranded region of bubble 15 was moved 20 bp closer to TATA in two different ways: (i) 20 bp of internal promoter sequence was deleted (bubble 15[Δ20]), such that the bubble 15 sequence was moved to the position occupied by the inactive bubble 10, with the sequence immediately upstream being the same as that in bubble 15, and (ii) the bubble 10 single-stranded DNA sequence was precisely replaced by that of bubble 15 (bubble 10::15). In contrast to the nearly inactive bubble 10 template, these two new variants promoted initiation although with less efficiency than that of bubble 15 (B, lanes 1, 3, and 5). The sequence upstream of the bubble contributed to the efficiency of transcription, since bubble 15[Δ20] was transcribed more efficiently than bubble 10::15 (B, lanes 3 and 5), although neither was transcribed as well as bubble 15. These results show that the sequence of the bubble is critical for transcription activity, but the position with respect to TATA and the upstream DNA sequence can also influence transcription efficiency. In agreement with the above-described finding that the sequence of the heteroduplex region determines the responsiveness to TFIIF, transcription from both of these new bubble variants was stimulated by TFIIF (B, lanes 5 to 8), consistent with the fact that their single-stranded DNA is identical to that of bubble 15, which is normally stimulated by TFIIF.
Fig 5 Promoter sequence and spacing affect initiation efficiency and the response to TFIIF. (A) Sequences of the HIS4 variants. Highlighted are the TATA box (in red), the human transcription start site position (in green), and S. cerevisiae start sites (in (more ...) A single-base change in the heteroduplex region alters the response to TFIIF.
To further test the finding that the DNA sequence of the bubble determines TFIIF responsiveness, we replaced the bubble 15 sequence with that of bubble 3 (bubble 15::3). As predicted, transcription from this new bubble was partially repressed by TFIIF (B, lanes 11 and 12). High-resolution analysis showed that TFIIF strongly repressed initiation within the bubble while slightly stimulating initiation in downstream double-stranded DNA, analogous to the behavior observed with bubble 3 (C, lanes 1 and 2).
The DNA sequences of bubbles 3 and 15 just upstream from the 3′ single-strand–double-strand junction are CTC (bubble 3) and CGC (bubble 15), where the G in bubble 15 (position +14) is the major initiation site in the presence of TFIIF. To test if this sequence difference is responsible for the different TFIIF responses, we altered bubble 3 base T at position −32 to G (A) and measured transcription with and without TFIIF. In comparison to bubble 3, where initiation within the single-stranded DNA was repressed by TFIIF, the single-base change in bubble 3 switched the response to TFIIF so that transcription from position −32 was now stimulated by TFIIF. Combined, our results show that the sequence of the heteroduplex near the single-strand–double-strand junction can have a strong influence on the response to TFIIF.
Fig 6 Sequence of the template strand dictates the TFIIF response and the transcription start site. (A) High-resolution analysis of initiation from bubble 3 and a variant promoter. The variant promoter contains a single-nucleotide change at position −32. (more ...) The template strand sequence determines the response to TFIIF.
In principle, the modulation of transcription by TFIIF could be in response to changes in the template or nontemplate strand or both strands of the heteroduplex. To test which DNA strands are responsible for the TFIIF response, four heteroduplex variants, diagramed in B, were tested for transcription with and without TFIIF. All these variants were created at the site of bubble 15 (B). We chose to pair the bubble 6 sequence with bubble 15, since these two sequences are not complementary. Bub15/Bub15 is identical to bubble 15, with the wild-type HIS4 sequence on the template strand and identical bases on the opposite nontemplate strand. The other variants have either the bubble 6 heteroduplex or bubble 15 and 6 on either the template or nontemplate strands, as diagramed.
C shows that initiation from bubble 15 at the G at position +14 is stimulated by TFIIF and that this pattern is the same when bubble 15 is only on the template strand (Bub6/Bub15) (lanes 2, 3, 8, and 9). In contrast, initiation from the single-stranded region of bubble 6 is repressed by TFIIF, with initiation starting primarily within the double-stranded region of the promoter. This behavior is identical to that observed when bubble 6 is present only on the template strand (Bub15/Bub6) (C, lanes 5, 6, 11, and 12). Also, note that the patterns of transcription initiation on all the templates with Pol II alone are nearly identical, but at a low level, compared to that when TBP and TFIIB are also present. Therefore, Pol II has an inherent preference for the initiation sites within the single-stranded bubbles that is enhanced by TBP and TFIIB. Together, our results demonstrate that it is the template strand that determines that transcription initiation pattern and the responsiveness to TFIIF.
A primary function of the TFIIB reader and linker regions is in initiation and/or stability of DNA melting.
The bubble templates allowed us to test the function of the TFIIB B-reader and B-linker regions. Previously, it was shown that mutations in the B-reader of the archaeal factor TFB were suppressed to various extents by the preopening of the DNA and that mutations in the B-linker were suppressed by archaeal TFE, which was proposed to function by stabilizing the DNA bubble (14
). Several mutations were generated in the TFIIB reader and linker regions and tested by using the complete reconstituted transcription system on double-stranded DNA (, lanes 1 to 5). These TFIIB mutations resulted in little or no transcription. In contrast, all of these mutations were almost fully suppressed by the heteroduplex templates bubble 3 and bubble 15 (, lanes 6 to 15). The fact that transcription from these templates is suppressed so efficiently by preopened DNA suggests that a primary function of the B-reader and B–linker regions is in the formation and/or stabilization of single-stranded DNA in the open complex state.
Fig 7 TFIIB B-reader and B-linker mutants are active in minimal open complexes. (A) In vitro transcription comparing wild-type (WT) TFIIB with B-reader/linker mutants. Shown are data for transcription utilizing a complete purified transcription system on double-stranded (more ...)
High-resolution analysis of initiation using the reader and linker mutants showed that they initiate from the same positions within single-stranded DNA as those of wild-type TFIIB; however, they initiate poorly from double-stranded DNA just downstream from the bubble. Transcription using the TFIIB reader mutants, like with wild-type TFIIB, is repressed by the addition of TFIIF. In contrast, transcription using the TFIIB linker mutant L110P is repressed by TFIIF for single-stranded initiation but is stimulated for initiation within downstream double-stranded DNA. This distinct behavior shows that the roles of the linker and reader are not identical in the responses to TFIIF.
Unexpected architecture of open complexes and role of TFIIF in TFIIB positioning.
An important question is how the architecture of the open complex differs from that of the PIC. To probe the structure of the minimal open complexes, TFIIB-FeBABE derivatives were used to form either PICs with double-stranded DNA and the complete reconstituted system or minimal open complexes with the bubble templates. The activation of FeBABE with H2
generates hydroxyl radicals that cut polypeptides within ~30 Å and allows the mapping of protein-protein interactions in large complexes (3
). The cleavage of the Pol II subunit Rpb1 was monitored by Western blotting using an antibody reactive against the N terminus of Rpb1. A (lanes 2 and 3) shows that in the PIC, FeBABE positioned within the TFIIB Zn ribbon at either residue 37 or 53 generates strong cleavage within the Rpb1 active site and dock region (A/D) and within the Rpb1 clamp domain, as previously shown (2
). The probing of open complexes formed on bubble 3 with these same TFIIB derivatives gave an identical pattern, showing that the positions of the Zn ribbon domain are similar in PICs and open complexes (A, lanes 8 and 9). Similarly, FeBABE positioned at TFIIB residues 67 and 118, in the B-reader and B-linker regions, respectively, gave similar cleavage patterns in both PICs and open complexes (A, lanes 4, 5, 10, and 11), showing that the reader and linker loops are positioned similarly in both complexes.
Fig 8 TFIIF positions the TFIIB core domain in the open complex. (A) TFIIB derivatives were conjugated to FeBABE at the indicated cysteine residues and assembled in PICs on a duplex HIS4 promoter (lanes 1 to 6) or in minimal open complexes on the bubble 3 promoter (more ...)
In contrast is the cleavage generated by FeBABE linked to the TFIIB core domain at residue 135 (green residue in D). In fully assembled PICs, this derivative generates strong cleavage in the Rpb1 clamp domain (A, lane 6, and D, blue highlighted surface) and in the fork/protrusion domain of Rpb2 (pink surface) (2
). Importantly, this strong Rpb1 cleavage is absent in the open complex (A, lane 12). These results suggest that while the TFIIB Zn ribbon and reader/linker regions are positioned on Pol II similarly in both the PIC and open complexes, the position of the TFIIB core domain is very different, with the TFIIB core domain in the minimal open complex positioned away from the Pol II wall domain.
These mapping results suggested that one of the other general factors is responsible for the positioning of the TFIIB core domain within the PIC. To test whether TFIIF contributes to TFIIB positioning, PICs or minimal open complexes were assembled with TFIIB-FeBABE (at residue 135) and with or without TFIIF (B). PICs assembled lacking TFIIF contained all added general factors and Pol II (not shown), likely due to the high concentrations of factors used for assembly. These incomplete PICs were not active in initiation from double-stranded DNA. Rpb1 cleavage was monitored by using an antibody reactive against the N terminus of Rpb1. These results show that Rpb1 cleavage is observed only when TFIIF is present.
To extend these findings, TFIIB derivatives with FeBABE at either residue 135 or 184 on the core domain were used to probe the cleavage of Rpb2 containing a triple-Flag tag at the C terminus (C and D). In complete PICs, FeBABE at residue 184 cleaves primarily the Rpb2 wall domain (brown surface in D), while FeBABE at position 135 cleaves the fork/protrusion. Rpb2 cleavage from both these FeBABE-labeled positions was observed only upon the addition of TFIIF (C, lanes 2 and 4). Together, these results show that the TFIIB core domain is positioned differently in the PIC and minimal open complexes and that TFIIF is primarily responsible for this difference.
TFIIF-dependent positioning of TFIIB contributes to repression of open complex activity.
Since TFIIF repressed transcription from many of the bubble templates and has a dramatic effect on the location of the TFIIB core domain, we tested if the TFIIF-dependent positioning of the core domain contributes to repression. To test this hypothesis, the positioning of the TFIIB core needed to be unlinked from the presence of TFIIF. Given the results presented above, we reasoned that the Zn ribbon and possibly the reader/linker would be necessary for the full activity of the open complexes but that the TFIIB core domain would be dispensable. To generate a construct lacking the TFIIB core domain, the N terminus of TFIIB containing the ribbon and reader/linker regions was fused to the N terminus of TBP (A). The first 60 residues of yeast TBP is not conserved and likely serves as a flexible linker between the TFIIB N terminus and the TBP conserved domain.
Fig 9 The TFIIB core domain is required for repression of open complexes by TFIIF. (A) Schematic diagram of full-length TBP, TFIIB, and the IIBN-TBP fusion protein. (B) In vitro transcription from the bubble 3 promoter by Pol II alone (lane 1), Pol II plus (more ...)
This recombinant factor was purified and, as expected, had no activity in the reconstituted transcription system with double-stranded DNA (not shown). In striking contrast, the TFIIBN-TBP fusion worked nearly as well to promote transcription from bubble 3 as did TBP and TFIIB (B, compare lanes 2 and 4). If the TFIIF-dependent positioning of the TFIIB core domain on Pol II contributes to repression, then transcription using the fusion construct lacking the core domain should be resistant to TFIIF. As predicted by this model, the addition of TFIIF had no repressive effect, in contrast to the system with TBP and TFIIB, which was repressed by TFIIF (B, lanes 4 and 5). High-resolution analysis showed that the TFIIB-TBP fusion allowed initiation at the same position in the single-stranded DNA bubble as that of wild-type factors, but the fusion was defective in promoting initiation from downstream double-stranded DNA (C). Together, our results suggest that the positioning of the TFIIB core domain on the Pol II wall at bubble 3 is inhibitory to initiation.