©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of cis-Acting Elements That Can Obviate a Requirement for the C-terminal Domain of RNA Polymerase II (*)

(Received for publication, November 7, 1994; and in revised form, December 21, 1994)

Andrew B. Buermeyer (§) Lee A. Strasheim Stephanie L. McMahon Peggy J. Farnham (¶)

From the McArdle Laboratory for Cancer Research, Medical School, University of Wisconsin, Madison, Wisconsin 53706

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have used an in vitro RNA polymerase II (RNAP II) inhibition-restimulation assay to investigate the inability of a form of RNAP II (RNAP IIB) that lacks the conserved, C-terminal heptapeptide repeat domain (CTD) to transcribe the dihydrofolate reductase (dhfr) promoter. Our previous studies demonstrated promoter-specific responses to RNAP IIB in the inhibition-restimulation assay and suggested the existence of cis-acting elements that alleviate the requirement for the CTD. We have now identified elements from two different classes of promoters that can convert dhfr to a CTD-independent promoter. First, addition of a consensus TATA box to the dhfr promoter resulted in a promoter capable of CTD-independent transcription and increased the promoter's affinity for the general transcription factor TFIID. These results suggest that high affinity for TFIID correlates with an ability to be transcribed by RNAP IIB, supporting a proposed interaction between the CTD and TFIID. Second, transfer of a combination of two elements (located at -25 and +1) from the rep-3b promoter, which does not contain a consensus TATA box but can nonetheless be transcribed by RNAP IIB, into the dhfr promoter also allowed CTD-independent transcription. These elements do not constitute a high affinity binding site for TFIID, indicating that an additional mechanism exists to allow CTD-independent transcription. Thus, elements from two classes of CTD-independent promoters that can obviate a requirement for the CTD appear to function via distinct mechanisms. Our finding that a change in a basal element can affect a requirement for the CTD is consistent with a role for the CTD during the formation of the transcriptional preinitiation complex.


INTRODUCTION

The C-terminal domain (CTD) (^1)of the largest subunit of RNA polymerase II (RNAP II) consists of multiple repeats of a heptapeptide sequence, the consensus for which is Tyr Ser Pro Thr Ser Pro Ser(1) . Although essential for cell viability(2, 3, 4, 5) , the exact role of the CTD in transcription remains unclear. In yeast cells, the CTD responds to signals from both transcriptional activators (6, 7) and repressors(8) . Also, phosphorylation of the CTD may play a role in the transition from initiation to productive elongation(9) . Formation of the preinitiation complex, containing RNAP II and the general transcription factors (required for the transcription of all RNAP II promoters), requires RNAP II containing an unphosphorylated CTD (RNAP IIA)(10, 11, 12) . However, elongation of transcripts is accomplished by RNAP II containing an extensively phosphorylated CTD (RNAP II0)(11, 13, 14) .

In contrast to the essential role of the CTD for cell viability, initial studies suggested that the CTD is not required for transcription in vitro. Several promoters can be transcribed in vitro by a form of RNAP II that lacks the CTD (RNAP IIB) (15, 16, 17, 18) . Although not thought to exist in cells, RNAP IIB can be purified by proteolytic removal of the CTD(1) . The in vitro results can be reconciled with the in vivo results with the suggestion that some promoters would require the CTD for transcription in vitro. In support of this, Thompson et al.(17) used a monoclonal antibody (mAb) that reacts with the CTD to inhibit transcription in vitro and then demonstrated that the dihydrofolate reductase (dhfr) promoter is active with RNAP IIA, but not RNAP IIB. We extended these results by demonstrating that several other promoters also displayed this CTD-dependent transcription in vitro(18) , indicating that the CTD plays a critical role in transcription from some, but not all, promoters. The result with the dhfr promoter has been confirmed by Kang and Dahmus(11) , who found that RNAP IIA, but not RNAP IIB, can restore activity to the dhfr promoter in whole cell extract fractions depleted of RNAP II activity by anion-exchange chromatography. The promoter-specific responses to RNAP IIB suggest the existence of cis-acting elements that influence a requirement for the CTD. We reasoned that identification and characterization of such elements would provide insight into the role of the CTD during transcription. Herein we describe the identification of two distinct elements that, when transferred into the dhfr promoter, allow CTD-independent transcription in vitro. Our results suggest that these elements function by distinct mechanisms, and are consistent with a proposed role for the CTD during the formation of the transcriptional preinitiation complex.


MATERIALS AND METHODS

DNA Constructs

Plasmids pBSST410 and pBSMM285 contain murine dhfr promoter sequences from -356 to +61 and from -270 to +17, respectively, in pBSM13plus (Stratagene, La Jolla, CA). The dhfr promoter with the E2F-binding site mutation has been described (mutant E, J in Means et al.(19) ). Plasmids containing murine rep-3b promoter sequences from -49 to +14 (pDHD62), from -192 to +14 (pDHS205), and sequences from the human H2b, human raf-1, murine interferon regulatory factor 1 (Irf1), hamster carbamoyl phosphate synthetase-aspartate transcarbamylase-dihydroorotase (CAD), and Drosophila hsp70 gene promoters have been described(18, 20, 21, 22) . Mutations of the rep-3b promoter at the RIP site, at -23/-22, and at +1/+14 were introduced by insertion of double-stranded oligonucleotides (McArdle oligonucleotide systhesis facility) spanning -25 to +14 into pDHD62 or pDHS205. To construct the dhfr+TATA promoter, mutations at -28, -26, -24, -23, and -22 were introduced into dhfr promoter DNA using a polymerase chain reaction assay (23) with a primer containing the appropriate mismatches. Plasmid pDR-5/+14 was created by ligating a fragment containing dhfr sequences from -356 to -17 to a fragment containing rep-3b sequences from -5 to +14, plus linker sequence extending to -16. Plasmids pDR-30/-18 and pDR-30/-18/-5/+14 were cloned in two steps. First double-stranded oligonucleotides (Genosys, The Woodlands, TX) containing hybrid promoter sequences from -38 to +14 were cloned into FspI-PstI-cut pBSStu(24) . Then, to ensure that the transcribed sequences were identical to pDR-5/+14, a PstI-AflIII fragment containing vector sequences from pBSM13plus was inserted into the same sites in the two hybrid promoters.

Purification of RNAP II and mAb 8WG16

RNAP IIA and IIB were purified from calf thymuses obtained from Pel-Freez Biologicals (Rogers, AR). RNAP IIB was prepared by the method of Hodo and Blatti (25) and dialyzed against transcription buffer D (26) as previously described(18) . RNAP IIA was prepared essentially as previously described (18) with modifications to be described elsewhere. (^2)RNAP IIA was diluted into transcription buffer D just prior to use. Monoclonal antibody 8WG16 was prepared as described elsewhere(18) .

Purification of TBP-containing Protein Fractions

Nuclear extract prepared from a HeLa cell line derivative LTRalpha3 (27) was fractionated on phosphocellulose P11 and DEAE-52 columns (Pharmacia Biotech Inc.) as previously described(28) . Protein was eluted off the DEAE column with a linear gradient of buffer D containing 0.05 to 0.9 M KCl. Consistent with previous descriptions(28) , the majority of TBP (as determined by Western blot analysis) eluted at approximately 0.3 M KCl. Protein fractions were frozen in liquid nitrogen and stored at -80 °C.

In Vitro Transcriptions

In vitro transcription reactions and the RNAP II inhibition-restimulation assay (18) (see Fig. 1) were performed using nuclear extract prepared from HeLa cells(18) . The results from all experiments were quantitated with a Betascope 2000 radioanalytical blot analyzer (Betagen, Waltham, MA) or a PhosphorImager 425S (Molecular Dynamics, Sunnyvale, CA) in accordance with manufacturer instructions.


Figure 1: Schematic of the RNAP II inhibition-restimulation assay. RNAP II activity in HeLa cell nuclear extract was inhibited by incubation with mAb 8WG16. Calf thymus RNAP IIA or RNAP IIB was added, and promoter activity was assessed in a standard in vitro transcription assay. RNA products were visualized either due to direct labeling during the transcription reaction (depicted above) using radiolabeled GTP (*GTP) or indirectly by primer extension analysis (24) of unlabeled RNA using a P-labeled primer.



Gel Mobility Shift Assays (GMSA)

Experiments measuring protein binding in nuclear extracts to the rep-3b promoter RIP element were performed essentially as described elsewhere(29) . Reactions (20 µl total volume) included 3 µg of nuclear extract prepared from HeLa cells, 2 µg of poly(dI-dC)bulletpoly(dI-dC), and 0.1 pmol of end-labeled probe (approximately 100,000 cpm). The RIP (5`-GATCCAGGCCTCGCAAGGTGGCGGCTTCTTCTG-3`) and RIPmt (5`-GATCCAGGCCTCGCAAACCTTCGCGAACTCGAG-3`) oligonucleotides contained promoter sequences from -23 to -5. Competitor DNAs were the unlabeled probes. Reactions containing everything except labeled probe were incubated for 15 min at room temperature, followed by the addition of the labeled probe and a second, identical incubation. The reactions then were electrophoresed for 2 h at 180 V (constant voltage) on a 4% (39:1) polyacrylamide gel with a 0.25 times TBE (23) running buffer. Prior to loading the samples, the gel had been prerun at 180 V for 15-30 min.

Detection of TBP-containing protein-DNA complexes was by a modification of the GMSA protocol described by Purnell and Gilmour(22) . The probe was a NruI-PvuII fragment containing sequences from -50 to +65 of the Drosophila hsp70 promoter. The probe fragment and specific competitor DNA fragments described below were isolated from nondenaturing, 4% polyacrylamide gels by incubating gel slices in an excess of Tris-EDTA (pH 7.6) overnight at 65 °C. Specific DNA competitors (115-130 bp) in the GMSA included: the unlabeled hsp70 probe, a fragment containing rep-3b promoter sequences from -192 to -81, and a fragment containing dhfr or dhfr+TATA promoter sequences from -64 to +61. Additional competitors (used in the experiment described in Fig. 9) were prepared by polymerase chain reaction and contained promoter sequences from -49 to +14 (rep-3b), -64 to +17 (dhfr), or -64 to +14 (pDR-30/-18, pDR-5/+14, and pDR-30/-18/-5/+14). These fragments contained additional vector DNA such that the fragments extended from approximately -65 to +90 (or to +120 for the dhfr competitor). Binding reactions (12.5 µl total volume) contained 0.5 µl of DEAE-52 protein fraction (approximately 1.5 µg of protein), 20,000-40,000 cpm end-labeled probe (approximately 4 ng), 100-200 ng of MspI-digested pBR322 DNA or HindIII-digested DNA (New England Biolabs), 0-200 ng of specific competitor DNA, 10 mM Hepes (pH 7.9), 5 mM MgCl(2), 90 mM KCl, 0.8 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 10% (v/v) glycerol. Supershift experiments also contained 130 ng of affinity-purified mAb, either TBP-18 that reacts with human TBP (^3)or NT73 that reacts with the beta` subunit of Escherichia coli RNAP(30) . All components of the reaction were mixed and incubated for 40 min at room temperature. The reactions then were electrophoresed for 4-5 h as previously described(22) .


Figure 9: Investigation of TFIID binding to the hybrid promoters. GMSA with the TFIID protein fraction and the hsp70 probe was performed as described in Fig. 6, except that these reactions contained 100 ng of MspI-digested pBR322 plasmid DNA as a nonspecific competitor. The position of the TBP-containing complex I is indicated. Binding to the probe was competed by the inclusion of specific competitor: either the hsp70 promoter fragment (lanes 2 and 3), or fragments from the indicated promoters including sequences from -49 to +14 (rep-3b), -64 to +17 (dhfr), and -64 to +14 (the hybrid promoters).




Figure 6: Investigation of TFIID binding to the dhfr and dhfr+TATA promoters. A, partially purified TFIID was incubated with end-labeled probes containing the hsp70 promoter sequences from -50 to +65 (lanes 1-14), the dhfr+TATA (lanes 15 and 16) or the dhfr wild-type (lanes 17 and 18) promoter sequences from -64 to +61. Reactions were electrophoresed on a native, 4% (80:1) polyacrylamide gel for 5 h at 4 °C. Three complexes resolved in the analysis are labeled I, II, and III. Binding to the hsp70 probe was competed by inclusion of unlabeled hsp70 (lanes 2-4), dhfr wild-type (lanes 8-10), or dhfr+TATA (lanes 11-13) promoter fragments. One additional competitor fragment (GC boxes) contained rep-3b promoter sequences from -192 to -81 (lanes 5-7). Reactions 15 and 16, and 17 and 18 are duplicates. B, GMSA with the TFIID fraction and the hsp70 probe were performed as above, except that the gel was run for approximately 10 h. Only the upper portion of the gel is shown, and the location of complex I is indicated. Reactions included no TFIID fraction (lanes 1 and 2), but did include mAb TBP-18 (alpha-TBP) (lane 1), that reacts with human TBP, or mAb NT73 (alpha-beta`) (lane 2), a mAb of the same isotype that reacts with the beta` subunit of E. coli RNAP. The remaining reactions received the TFIID fraction (lanes 3-8) and either no mAb (lanes 3 and 8), mAb TBP-18 (lanes 4 and 5), or mAb NT73 (lanes 6 and 7). Duplicate reactions are in lanes 3 and 8, 4 and 5, and 6 and 7.




RESULTS

We have used a RNAP II inhibition-restimulation assay (Fig. 1) (18) to investigate promoter-specific responses to RNAP II that lacks the CTD (RNAP IIB). RNAP II activity in nuclear extract was inhibited by incubation with a mAb that reacts with the CTD. The specificity of the inhibition of in vitro transcription is demonstrated by the ability to restimulate inhibited transcription by the addition of RNAP IIA(17, 18) . Transcription from all wild-type promoters that we have tested is restimulated by RNAP IIA (18) . However, RNAP IIB restimulates inhibited transcription only from a subset of promoters(17, 18) , indicating that the CTD plays an essential role in the in vitro transcription from these promoters. In our previous work(18) , we contrasted the response of the dhfr and rep-3b promoters to RNAP IIB in the inhibition-restimulation assay; both promoters are Sp1-activated (29) (data not shown) and lack a consensus TATA box 30 bp upstream of the start sites of transcription. However, unlike the dhfr promoter, the rep-3b promoter can be transcribed by RNAP IIB (18) . The sequences contained in the approximately 65-bp minimal promoter regions of the dhfr and rep-3b promoters determine the response of each of these promoters to RNAP IIB(18) . We postulated that protein binding to distinct sites, the HIP site in dhfr and the RIP site in rep-3b, within each minimal promoter sequence is responsible for the difference in the ability to be transcribed by RNAP IIB. To test this hypothesis, we needed to identify mutations in the HIP and RIP sites that block the binding of proteins present in transcription extracts.

Analysis of the Requirement for Protein Binding to the HIP and RIP Sites for Wild-type Promoter Response in the RNAP II Inhibition-Restimulation Assay

Recent work has demonstrated that the HIP site is a binding site for the E2F family of transcription factors (31, 32) and is necessary for a regulated increase in transcription from the dhfr promoter following serum-stimulation of quiescent NIH 3T3 cells(19, 33) . Mutations that block E2F binding to the HIP site have been described(19) . To analyze protein binding to the RIP site in the rep-3b promoter, gel mobility shift experiments (GMSA) were performed with HeLa cell nuclear extract. Using a probe containing rep-3b sequences from -23 to -5, we confirmed earlier findings (34) that specific protein-DNA complexes can form on this region of the promoter (Fig. 2). We mutated the region protected in the previous DNase footprinting assays and then determined that protein binding to the RIP site probe was not blocked by an excess of RIPmt oligonucleotide (Fig. 2; see also Fig. 3A for the sequence of the RIP site mutation). We next compared the ability of RNAP IIB to restimulate mAb 8WG16-inhibited transcription from wild-type and mutated dhfr and rep-3b promoters (Fig. 3). Mutation of the HIP (E2Fmt) and RIP sites did not affect significantly the level, or the location of the start sites of uninhibited transcription relative to that from the wild-type dhfr and rep-3b promoters (Fig. 3B), demonstrating that protein binding to these sites in the dhfr and rep-3b promoters is not necessary for the formation of competent transcription complexes. Additionally, neither mutation affected the response of the promoter to RNAP IIB in the inhibition-restimulation assay. Similar to the response of the wild-type promoter, transcription from the dhfr promoter with a mutated HIP (E2Fmt) site was not restimulated by addition of RNAP IIB (Fig. 3B). Transcription from both the wild-type rep-3b promoter and from the promoter with a mutated RIP site was restimulated by the RNAP IIB (Fig. 3B). These results demonstrate that binding of these factors to the sites in the dhfr and rep-3b promoters is not necessary for the promoter's response to RNAP IIB in the inhibition-restimulation assay.


Figure 2: Protein binding to the RIP site. Protein binding to the RIP site from the rep-3b promoter was examined in GMSA with HeLa cell nuclear extracts (no protein was added to lane 1). Protein binding to the probe was competed by prior incubation of the extract with unlabeled RIP (lanes 3-5) or RIPmt (lane 6) oligonucleotides (comp.). See Fig. 3A for the sequence of the RIP site mutation. The position of uncomplexed probe is indicated (unbound).




Figure 3: Mutation of the RIP site in rep-3b and the E2F site in dhfr has no effect in the inhibition/restimulation assay. A, sequences of the wild-type and mutant promoters from -32 to +10. Mutated sequences are indicated in bold, lowercase type. Promoter sequences included in the DNA templates are indicated in parentheses. The underlines indicate the consensus E2F binding site in the dhfr promoter and RIP site in the rep-3b promoter (as defined by the extent of the footprinted region determined previously(34) . B, response of the wild-type and mutated promoters to RNAP IIB in a RNAP II inhibition/restimulation assay. Uninhibited reactions (U) for each promoter are in lanes 1, 4, 7, and 10. Reactions in lanes 2, 5, 8, and 11 were inhibited with 150 µg/ml of mAb 8WG16 (I). Reactions in lanes 3, 6, 9, and 12 received the mAb and 500 ng of RNAP IIB (+pIIB). RNAs were visualized by primer extension analysis. The size of individual marker bands (M) for each panel are indicated. The signal in lane 11 appears lower than that in lane 8 due to loss of sample during processing. In other experiments, the wild-type rep-3b and the rep-3b RIPmt promoters responded similarly to the mAb 8WG16.



The Role for a Consensus TATA Box in a Promoter's Response to RNAP IIB in the Inhibition-Restimulation Assay

Previously, we reported that the presence of a consensus TATA box in a promoter is not sufficient to allow transcription by RNAP IIB. This conclusion was based on our finding that transcription from the human histone H2b promoter, although it contains a consensus TATA box, was not restimulated by RNAP IIB in the inhibition-restimulation assay(18) . However, in recent experiments, RNAP IIB could restimulate transcription from the H2b promoter (Fig. 4). We have attempted to reconcile the difference between our published and current results by testing the response of the H2b promoter in the inhibition-restimulation assay with multiple preparations of mAb 8WG16, RNAP IIB, DNA template, and nuclear extract. In each experiment, inhibited transcription from the H2b promoter was restimulated with RNAP IIB (data not shown). Additionally, we retested several other promoters from our previous analysis in the RNAP II inhibition-restimulation assay. As reported, transcription from the CAD and Raf-1 promoters, previously classified as nonresponsive to RNAP IIB (18) , was not restimulated by RNAP IIB (Fig. 4). These promoters do not contain a consensus TATA box(18) . Also as reported, RNAP IIB did restimulate transcription from the Irf1 and c-myc promoters (Fig. 4, and data not shown), previously classified as responsive to RNAP IIB(18) . The c-myc, but not the Irf1, promoter contains a consensus TATA box(18) . Thus, the response to RNAP IIB of all the retested promoters, except the H2b promoter, remained as published. Although we were unable to identify the cause of the difference in our results with the H2b promoter, we cannot rule out that a specific inhibitor of H2b was introduced during previous experiments or during manipulations of previous preparations of mAb or RNAP II. Those preparations no longer exist. Given the current results, we conclude that the H2b promoter can be transcribed by RNAP IIB.


Figure 4: The H2b promoter is capable of CTD-independent transcription. Responses of a panel of promoters to RNAP IIB in the inhibition-restimulation assay was reexamined. The labeling of individual lanes is as for Fig. 3B. Promoter sequences included in each template were as follows: -356 to +14 for the murine dhfr promoter, -175 to +230 for the human H2b promoter, -81 to +26 for the hamster carbamoyl phosphate synthetase-aspartate transcarbamylase-dihydroorotase promoter (CAD), -650 to +240 for the human raf-1 promoter, and -299 to +225 for the murine interferon regulatory factor gene 1 promoter (Irf1). Appropriate reactions received 160 µg/ml mAb 8WG16 and 500 ng of RNAP IIB. RNAs were visualized by direct labeling during the transcription reaction.



Although the presence of a consensus TATA box in a promoter is not required for restimulation by RNAP IIB in our assay ( Fig. 3and Fig. 4) (18) , all of the promoters tested that contain a consensus TATA box were transcribed by this form of RNAP II. We postulated, therefore, that the presence of a consensus TATA box in a promoter would be sufficient to allow transcription in the absence of the CTD. To test this hypothesis, we created a mutant dhfr promoter (dhfr+TATA), containing a consensus TATA box located 30 bp upstream of the wild-type start sites of transcription (Fig. 5A). In uninhibited extracts, the transcriptional activity of this promoter was approximately 3-fold greater than the wild-type dhfr promoter. Initiation utilized the same sites as the wild-type promoter, although transcription at a downstream start (at approximately +12; see Fig. 8A for the sequence in that region) was increased. We have not investigated the differential utilization of the start sites by these promoters, because, for each promoter, both start sites behaved similarly in the inhibition-restimulation assay (see below). In the inhibition-restimulation assay, transcription from the dhfr+TATA mutant promoter, unlike the response of the wild-type promoter, was restimulated by RNAP IIB (Fig. 5B). The response of both promoters to RNAP IIA was essentially the same (restimulation of 4-5-fold over the level of inhibited transcription) (Fig. 5B). Therefore, the addition of a consensus TATA box to the dhfr promoter is sufficient to allow transcription in the absence of the CTD.


Figure 5: Response of the dhfr and dhfr+TATA promoters to RNAP IIB in the inhibition-restimulation assay. A, sequences of the dhfr wild-type and dhfr+TATA promoters from -32 to +10. Promoter sequences included in the DNA templates are indicated in parentheses. The core of the consensus TATA box is indicated in bold type, and mutated sequences are indicated in bold, lowercase type. B, response of the wild-type and mutant promoters to RNAP IIB and RNAP IIA in an inhibition-restimulation assay. The labeling of individual lanes is as for Fig. 3B. Appropriate reactions received 105 µg/ml mAb 8WG16 and 500 ng of RNAP IIB. Reactions in lanes 4 and 8 received the mAb and 900 ng of RNAP IIA (+pIIA). RNAs were visualized by primer extension analysis.




Figure 8: Response of dhfr-rep-3b hybrid promoters to RNAP IIB in the inhibition-restimulation assay. A, sequences of wild-type dhfr (plain type) and rep-3b (bold type) promoters, and the dhfr-rep-3b hybrid promoters from -34 to +14 (+17 for dhfr). In the hybrid promoters (DR-30/-18, DR-5/+14, and DR-30/-18/-5/+14), rep-3b sequences transferred into the dhfr promoter are indicated in bold type. The lowercase type in DR-5/+14 (from -16 to -5) refers to linker DNA not included in either wild-type promoter (see ``Materials and Methods'' for details). The 5` end point of the promoter constructs are indicated in parentheses. The dhfr promoter construct is not precisely matched to the hybrid promoters since it includes less upstream dhfr sequences and some additional vector sequences just downstream (+18 to +51) of the promoter. Therefore, comparison of the transcriptional activity of the dhfr to the hybrid promoters is not appropriate. B, response of the wild-type and hybrid promoters to RNAP IIB in an inhibition-restimulation assay. The labeling of individual lanes is as for Fig. 3B. Appropriate reactions received 250 µg/ml of mAb 8WG16 and 500 ng of RNAP IIB. RNAs were visualized by primer extension analysis.



The consensus TATA box is a binding site for the TBP component of the general transcription factor TFIID, which additionally contains TBP-associated factors (TAFs)(27) . TAFs also contact the promoter DNA around and downstream of the start site of transcription, and these contacts contribute to the affinity of TFIID for the promoter(22, 35, 36, 37) . The complexity of the interactions between TFIID and the promoter DNA suggests that high affinity binding of TFIID cannot be predicted by sequence inspection alone. For example, in a promoter without a consensus TATA box, contacts made by TAFs might be sufficient to allow high affinity binding of TFIID. The affinity of TFIID for the dhfr promoter has never been examined. Therefore, we performed GMSA to assess the affinity of the dhfr and dhfr+TATA promoters for TFIID. In particular, we wished to determine if the ability of the consensus TATA box to obviate the dhfr promoter's CTD requirement might be due to an increased affinity of TFIID for the mutant promoter.

A TBP-containing fraction purified from HeLa cell nuclear extract was used in a GMSA with a probe containing sequences from -50 to +65 of the consensus TATA box-containing, Drosophila hsp70 promoter used previously to detect TFIID complexes in similar GMSA experiments (22) . Three complexes were detected on nondenaturing polyacrylamide gels (Fig. 6A). No complexes were formed in the absence of the TFIID fraction (Fig. 6B). The formation of the fastest mobility complex (complex III) was blocked by the addition of all competitor DNAs tested (Fig. 6A) and therefore was considered nonspecific. The middle complex (complex II) was not reproducible. The formation of the lowest mobility complex (complex I) was blocked by the inclusion of an excess of unlabeled probe, but not by a similar excess of a DNA fragment (GC boxes) containing the consensus Sp1 binding sites from the rep-3b promoter, sequences from -192 to -81 (Fig. 6A), suggesting that this complex represents binding to the probe specific for consensus TATA box-containing promoter DNA. The low mobility of this complex in these highly porous gels (4% polyacrylamide; 80:1) suggests that the complex has a high molecular weight. In addition, the mobility of complex I was retarded by the addition of a mAb that reacts with TBP (alpha-TBP), but not by the addition of a control mAb (alpha-beta`) (Fig. 6B). We conclude that the GMSA assay allows detection of a low mobility complex containing TBP that is bound specifically to a consensus TATA box-containing core promoter. By comparison with similar experiments done with a protein fraction isolated from Drosophila cells (22) we believe that complex I contains TFIID.

Affinity of the TBP-containing complex for the wild-type and mutant dhfr promoters was assessed in the GMSA by measuring the relative abilities of the two promoters to act as competitors. In competition experiments, the dhfr promoter (containing sequences from -64 to +61) did not reduce significantly the amount of TBP-containing complex seen (maximally, a 50% reduction relative to the level of uncompeted complex) (Fig. 6A). However, addition of the dhfr+TATA mutant promoter fragment did block formation of the complex (Fig. 6A). Comparison of the relative efficiency with which the fragments blocked the formation of complex I suggests that the addition of a consensus TATA box to the dhfr promoter results in a 4-5-fold higher affinity for the TBP-containing complex. The increased affinity of the dhfr+TATA promoter for the TBP-containing complex correlates with the higher transcriptional activity of this promoter (Fig. 5). In direct binding experiments, a specific complex that comigrated with that formed on the hsp70 promoter, and whose mobility was retarded specifically by the anti-TBP mAb (data not shown) was seen only with the dhfr+TATA promoter probe (Fig. 6A). We were unable to quantitate the direct binding experiments due to variable amounts of background smearing originating from complex II. However, our results suggest that addition of a consensus TATA box to the dhfr promoter results in a higher affinity for TFIID and, in addition, confers to the promoter an ability to be transcribed by RNAP IIB.

Investigation of rep-3b Promoter Elements That Influence the Response to RNAP IIB in the Inhibition-Restimulation Assay

The results of our investigations of the dhfr and dhfr+TATA promoters suggest that affinity of TFIID for any promoter would correlate with the ability to be transcribed by RNAP IIB. However, we have identified promoters (rep-3b and Irf1) that lack an apparent TATA box but nonetheless are transcribed by RNAP IIB in the inhibition-restimulation assay ( Fig. 3and Fig. 4)(18) . These promoters might contain a high affinity, nonconsensus TFIID-binding site responsible for their ability to be transcribed by RNAP IIB. We predicted that such a site would be required for high levels of transcription. Therefore, we identified elements in the rep-3b promoter necessary for efficient transcription in vitro. We focused our analysis on the sequences from -49 to +14, because this region of the promoter is sufficient to allow transcription by RNAP IIB(18) . The Sp1 site located at -41 was excluded from our analysis since both the dhfr and rep-3b promoters are activated by Sp1 in vitro(29) (data not shown) and since Sp1 can activate transcription driven by RNAP IIB(38) . Because mutation of the sequences from -21 to -6 (the RIPmt) did not affect the ability of the rep-3b promoter to be transcribed by RNAP IIB (Fig. 3), we reasoned that sequences between the Sp1 site and the RIP site (from -41 to -21), or sequences downstream of the RIP site (from -5 to +14) might be responsible for allowing transcription by RNAP IIB. We introduced mutations into these two regions of the rep-3b promoter and measured the effect on transcriptional activity in vitro. Mutation of a sequence located at -23 from AA to gc, or substitution of the sequences from +1 to +14 (Fig. 7A) resulted in a 70-90% reduction of in vitro transcriptional activity, relative to that of the wild-type promoter, demonstrating that mutation of these two sequences in the rep-3b promoter disrupts elements that are necessary for efficient activity in extracts. The ability of these two mutant promoters to be transcribed by RNAP IIA and RNAP IIB was assessed in the inhibition-restimulation assay. Transcription from the wild-type promoter was restimulated by both RNAP IIA and RNAP IIB. However, we were unable to detect any reproducible restimulation of transcription from the mutant promoters by either form of RNAP II. This might reflect the low, close to background activity of the mutant promoters. Alternatively, inability to restimulate transcription might reflect a defect in recruitment of either form of RNAP II to the mutant promoters.


Figure 7: Identification of critical elements in the rep-3b promoter. A, sequences of the wild-type and mutant rep-3b promoters from -34 to +14. Mutated sequences are indicated with bold, lowercase type. Each construct contained promoter sequences from -192 to +14. The average transcriptional activity (calculated from multiple experiments) of the mutant promoters relative to that of the wild-type promoter is listed (Activity). The -23/-22 mutant initiated from the same sites as the wild-type promoter; however, the +1/+14 mutant initiated at a slightly different position since the sequence at the start site of transcription was changed. B, response of the wild-type and mutant promoters to RNAP IIB and RNAP IIA in an inhibition-restimulation assay. The labeling of individual lanes is as for Fig. 5B. Appropriate reactions received 150 µg/ml of mAb 8WG16, 500 ng of RNAP IIB, and 900 ng of RNAP IIA. RNAs were visualized by primer extension analysis. The transcriptional activity of both mutant promoters in uninhibited extracts in the experiment shown was approximately 20% of the activity of the wild-type promoter.



Since we were unable to determine whether the elements identified in the mutagenesis of the rep-3b promoter were necessary for activity with RNAP IIB, we sought to determine if these elements, when transferred into the dhfr promoter, would be sufficient to allow transcription by RNAP IIB. We created the hybrid dhfr-rep-3b promoters, DR-30/-18, DR-5/+14, and DR-30/-18/-5/+14 by substituting rep-3b sequences from -30 to -18, or from -5 to +14, or both sets of rep-3b sequences, respectively, for the analogous dhfr sequences (Fig. 8A). The hybrid promoters otherwise contain dhfr sequences from -356 to +14. The transcriptional activity and start site pattern of these promoters was determined by primer extension analysis of in vitro produced RNAs (see Fig. 8B for examples). Transcription from DR-5/+14 and DR-30/-18/-5/+14 initiated predominantly from +1 (Fig. 8B). For DR-30/-18, the predominant starts mapped to approximately -6 (Fig. 8B), even though this promoter contains the wild-type dhfr start sites. The hybrid promoters did not have significantly higher activity than a dhfr promoter containing sequences from -270 to +17 (Fig. 8B). However, a direct comparison of transcriptional activity is not appropriate because this particular dhfr promoter construct is not matched precisely to the hybrid promoters (see Fig. 8legend for details). Importantly, the activity of the hybrid promoters was high enough to allow analysis in the inhibition-restimulation assay. Transcription from the dhfr, the DR-30/-18, and the DR-5/+14 promoters was not restimulated significantly (maximally 1.3-fold) by RNAP IIB, indicating that neither single substitution of rep-3b sequences is sufficient to allow transcription from the dhfr promoter by RNAP IIB (Fig. 8B). However, transcription from DR-30/-18/-5/+14 and the wild-type rep-3b promoters was restimulated similarly by RNAP IIB (Fig. 8B), demonstrating that the double substitution of rep-3b sequences into the dhfr promoter resulted in a promoter capable of CTD-independent transcription.

Because TFIID recognizes not only the TATA box, but also the sequences around the start site of transcription(35, 37) , the rep-3b sequences from -30 to -18 and from -5 to +14 might constitute a TFIID-binding site. Therefore, we assessed the relative ability of the rep-3b, the dhfr, and the dhfr-rep-3b hybrid promoters to act as competitors in the GMSA with the partially purified TFIID fraction. 100 or 200 ng of each competitor were included in binding reactions containing the hsp70 core promoter and the partially purified TFIID fraction. In control reactions, inclusion of only 50 ng of the unlabeled hsp70 probe reduced the level of complex I by 80% (Fig. 9). In contrast the rep-3b, dhfr, and dhfr-rep-3b hybrid promoters all displayed a similar, inefficient ability to compete, maximally reducing the level of complex detected by 40% of the level of uninhibited complex (Fig. 9). The inability to compete efficiently for formation of the TBP-containing complex suggests that these promoters all have similar low affinities for TFIID. Therefore, the ability of the rep-3b and the DR-30/-18/-5/+14 promoters to be transcribed by RNAP IIB does not correlate with a higher affinity for TFIID.


DISCUSSION

We have used an RNAP II inhibition-restimulation assay to investigate the inability of the dhfr promoter to be transcribed by RNAP IIB. Mutation of an E2F binding site located at +1 in the dhfr promoter did not allow the promoter to be transcribed by RNAP IIB, indicating that E2F-mediated repression was not preventing transcription by RNAP IIB. However, addition of a consensus TATA box to the dhfr promoter obviated the requirement for the CTD. This same mutation increased the affinity for a TBP-containing complex, suggesting that high affinity for TFIID (provided by a consensus TATA box) correlates with an ability to be transcribed by RNAP IIB. This hypothesis is supported by our findings that all promoters that we have tested containing a consensus TATA box were transcribed by RNAP IIB in the inhibition-restimulation assay. However, we also have identified promoters that do not contain a consensus TATA box, but that nonetheless are transcribed by RNAP IIB in the inhibition-restimulation assay. In investigations of one such promoter, the rep-3b promoter, we have identified two mutations that disrupt elements essential for efficient transcription in vitro. Transfer of a combination of these elements, but not either element alone, into the dhfr promoter (to create the hybrid promoter DR-30/-18/-5/+14) obviated the requirement for the CTD for transcription. However, although these elements are located at approximately -30 and around the start site of transcription, they apparently do not constitute a nonconsensus, high affinity binding site for TFIID, as determined by the inability of the rep-3b or the DR-30/-18/-5/+14 promoters to act as efficient competitors in GMSA with a partially purified TFIID fraction.

Transcription is a multistep process and it is of interest to understand at what step the CTD is required for transcription from the dhfr promoter. For example, RNAP IIB might enter into a stable preinitiation complex at the dhfr promoter, but this complex might be unable to initiate transcription, or might be defective for elongation of transcription. Detection of the dhfr promoter's transcription products in our experiments requires an RNA of at least 60 nucleotides, and therefore we have not determined at what point RNAP IIB fails in transcription from this promoter. However, our finding that a change in a basal element (the TATA box) changes a requirement for the CTD suggests that the CTD is required during the formation of the preinitiation complex. This model is supported by the results of experiments in which the mAb 8WG16 inhibited transcription only when added prior to the formation of preinitiation complexes(39) . In addition, excess RNAP IIB does not inhibit transcription from the dhfr promoter directed by a limiting amount of RNAP IIA, suggesting that RNAP IIB does not enter into stable, nonfunctional preinitiation complexes at this promoter (11) . Thus, in vitro, the CTD apparently is required during the formation of the preinitiation complex.

We have proposed that high affinity for TFIID correlates with an ability to be transcribed by RNAP IIB, suggesting that increased occupancy of the promoter by TFIID obviates a requirement for the CTD. This model is consistent with a proposed functional interaction between TFIID and the CTD. Several groups have suggested that the CTD and TFIID (and/or TBP) interact during the formation of the preinitiation complex (39, 40) . For example, synthetic peptides consisting of several consensus heptapeptide repeats from the CTD can bind directly to TBP (40) . Although the mechanism of the formation of the preinitiation complex at promoters like dhfr that do not contain a consensus TATA box is unknown, a possible role for a CTD-TFIID interaction is depicted in Fig. 10. TFIID is recruited to the promoter via interactions with Sp1(35, 41, 42, 43, 44) , a factor required for transcription from the dhfr promoter in crude extracts(29) . RNAP II, found in solution bound to TFIIF and TFIIB (45, 46, 47, 48) , is recruited to the promoter via a combination of interactions (indicated by the double-headed arrows) that are either CTD-independent or -dependent (Fig. 10A). Successful recruitment of RNAP II into a stable complex at the dhfr promoter requires both types of interactions, such that removal of the CTD prevents the formation of productive complexes (Fig. 10B). A promoter with a consensus TATA box, and therefore high affinity for TFIID, can recruit RNAP II via the CTD-independent interactions alone, and therefore no longer requires the CTD (Fig. 10C).


Figure 10: A possible role for CTD-TFIID interactions during formation of the preinitiation complex. A, the transcriptional activator Sp1, required for dhfr promoter activity in crude extracts(29) , recruits the general transcription factor TFIID to the dhfr promoter (solid line). RNAP II, shown in a complex with the general transcription factors TFIIB and TFIIF, is recruited to the promoter via a combination of interactions (double-headed arrows) that are CTD-independent and -dependent. Other general transcription factors that enter the complex after RNAP II are not depicted for sake of simplicity. B, CTD-independent interactions between TFIID and the RNAP II complex are not sufficient to allow the formation of a stable complex including RNAP IIB at the dhfr promoter. C, inclusion of a consensus TATA box (TATA) to the dhfr promoter increases the affinity of the promoter for TFIID and allows the formation of a productive complex in the absence of the CTD.



Based on the model described in Fig. 10, one might be led to predict that mutation of a consensus TATA box in a CTD-independent promoter would result in a reduction in the ability of RNAP IIB to transcribe the promoter. However, analysis of a given promoter in such a manner could be complicated by the presence of redundant elements. For example, if more than one protein binds to the promoter that can contribute to the recruitment of RNAP II via CTD-independent interactions, then mutation of any one element would not demonstrate a reduction in the ability to be transcribed by RNAP IIB. Indeed, the existence of elements other than TATA boxes that can confer the ability to be transcribed by RNAP IIB is demonstrated by the results of our investigations with the rep-3b promoter. Elements located at approximately -25 and +1 in the rep-3b promoter, when inserted to replace the analogous sequences in the dhfr promoter, confer CTD-independent transcription to the dhfr promoter. However, this hybrid promoter does not have an increased affinity for TFIID, relative to that of the dhfr promoter. The elements in the rep-3b promoter might function as a binding site for an activator that recruits TFIID to the promoter, or that directly or indirectly recruits RNAP II via interactions that are CTD-independent. To date, however, we have not identified protein binding specifically to these sequences of the rep-3b promoter (data not shown). This raises the possibility that a second mechanism exists to allow CTD-independent transcription. For example, the rep-3b elements might allow the promoter to adopt a particular structural conformation that is necessary for the formation of stable preinitiation complexes. The dhfr promoter sequences might require the CTD to adopt the same conformation, raising the possibility that the CTD interacts directly with the promoter DNA. This model is supported by findings that synthetic CTD peptides can bind to, and change the structure of DNA, possibly by partial intercalation between the bases(49, 50) . However, the identified rep-3b elements are not obviously more A + T-rich than the analogous dhfr sequences such as to be predicted to denature or bend more easily. Understanding the function of these elements in the rep-3b promoter will require more investigation.

Our characterization of DNA elements that influence the in vitro requirement for the CTD represent an important step in understanding the function of this domain of RNAP II. Although it is likely that the CTD has muliple functions in vivo, a necessary role in the formation of preinitiation complexes at the promoters of essential genes, like the dhfr gene, may partially explain the essential nature of this domain.


FOOTNOTES

*
This work was supported by United States Public Health Service Grants CA45240 and CA59524 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by United States Public Health Service Training Grant GM07215 from the National Institutes of Health.

To whom correspondence should be addressed: McArdle Laboratory, 1400 University Ave., Madison, WI 53706. Tel.: 608-262-2071; Fax: 608-262-2824.

(^1)
The abbreviations used are: CTD, C-terminal heptapeptide repeat domain; RNAP, RNA polymerase; mAb, monoclonal antibody; CAD, carbamoyl phosphate synthetase-aspartate transcarbamylase-dihydroorotase; GMSA, gel mobility shift assay; TF, transcription factor; TBP, TATA-binding protein; TAF, TBP-associated factor; HIP, housekeeping initiation protein; Irf, interferon regulatory factor; bp, base pair(s).

(^2)
L. A. Strasheim and R. R. Burgess, unpublished data.

(^3)
N. E. Thompson and R. R. Burgess, unpublished data.


ACKNOWLEDGEMENTS

For the generous gift of various reagents, we thank Richard Kraus and Janet E. Mertz (partially purified TFIID), Nancy E. Thompson and Richard R. Burgess (mAbs to TBP and beta`), and David L. Gilmour (hsp70 promoter plasmid and GMSA protocol). Additionally, we thank Chris R. Bartley, Dave G. Richards, and Kris Sukow for technical assistance, and members of the Farnham laboratory for discussion and comments on this manuscript.


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