roles of tata and initiator elements in determining the start site

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 2, Issue of January pp. 1391-1402,1992 Printed in U. S. A.

Roles of TATA and Initiator Elements in Determining the Start Site Location and Direction of RNA Polymerase I1 Transcription*

(Received for publication, June 10, 1991)

Anne O’Shea-Greenfield and Stephen T. SmaleS From the Howard Hughes Medical Institute, Molecular Biology Institute, and Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, California 90024

Transcriptional initiator elements and TATA elements are functionally similar, in that both can act in concert with a Spl-dependent activator element to direct high levels of accurately initiated transcription. In this study, we have used in vitro transcription experiments to elucidate the relative activities of TATA and initiator. By varying the locations of TATA and initiator elements within a simple promoter, we compared their abilities to localize transcription start sites and to mediate transcriptional activation by Spl. In addition, we addressed the contributions of initiator and TATA towards determining the direction of transcription from a promoter. Finally, we addressed the prevalence of initiator elements by analyzing the start site regions of several genes. We found that many of these regions possess initiator activity, although an initiator consensus sequence could not be defined. Taken together, the data presented suggest that an initiator is a common element that can influence the direction of transcription as well as the location of a transcription start site. However, in general, an initiator was incapable of altering these activities of a TATA box within a promoter containing both core elements.

Accurate transcription from many protein-coding genes depends on the presence of a TATA box (reviewed in Breath- nach and Chambon, 1981). In larger eukaryotes, this element directs the formation of a stable preinitiation complex, in which RNA polymerase I1 is poised to begin transcription at a distance from TATA of 25-30 nucleotides (reviewed in Sawadogo and Sentenac, 1990). The preinitiation complex is nucleated by the binding to TATA of a sequence-specific DNA-binding protein called TFIID.’ Following TFIID binding, TFIIB, TFIIF, RNA polymerase 11, and finally TFIIE are incorporated to form the complete complex. Transcriptional activation by proteins binding to upstream promoter sequences often requires an intact TATA box, and some activator proteins appear to influence the stability or formation

* This work was supported by the Howard Hughes Medical Insti- tute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$To whom correspondence should he addressed. Tel.: 213-206- 4777; Fax: 213-206-5344.

’ The abbreviations used are: TFIID, B, E, F, transcription factors IID, IIB, IIE, IIF; SV40, simian virus 40; DHFR, dihydrofolate reductase; Inr, initiator element with homology to and including the TdT initiator; Ad, adenovirus; AdML, adenovirus major late; AMP-PNP, adenyl-5”yl imidodiphosphate; bp, base pair; TdT, ter- minal deoxynucleotidyltransferase.

rate of the preinitiation complex (Sawadogo and Roeder, 1985; Horikoshi et al., 1988a, 198813; Lin and Green, 1991).

In addition to its roles of determining the transcription start site, directing the formation of a preinitiation complex, and mediating the activity of upstream activator elements, the TATA element is thought to be responsible for determining the direction along the DNA template in which transcription proceeds. This role for TATA is supported by the observation that some TATA elements are largely unidirectional. Reversing the orientation of this element within the context of an intact promoter has been found to dramatically reduce promoter strength (Nagawa and Fink, 1985; Bielinska et al., 1989). Furthermore, formation of a stable preinitiation complex over an isolated TATA box occurs in a directional manner (Sawadogo and Roeder, 1985; Nakajima et al., 1988; Van Dyke et al., 1988; Buratowski et al., 1989). In contrast to the unidirectional nature of TATA, most activator elements are bidirectional (reviewed in Johnson and McKnight, 1989), and several have been found downstream from transcription start sites (e.g., Biggin and Tjian, 1988; Soeller et al., 1988; Perkins et al., 1988). This flexibility of activator elements provides further support for the notion that a component of the core promoter region must determine the direction of RNA synthesis.

Although the above arguments suggest that the most ob- vious determinant of directionality is the orientation of a largely unidirectional TATA box, the issue of transcriptional directionality and the involvement of TATA remains unre- solved and untested. Many promoters do not appear to contain TATA elements (e.g. Ayer and Dynan, 1988; Sehgal et al., 1988; Smale and Baltimore, 1989; Means and Farnham, 1990; and references therein), and many different AT-rich elements can functionally interact with TFIID (Hahn et al., 1989). Some of these elements appear to be symmetrical, suggesting that they may not be unidirectional. Moreover, it has not been demonstrated that reversal of a consensus TATA element in a natural promoter activates transcription in the opposite direction as it decreases transcription in the correct direction. Thus, the orientation of TATA may have little or nothing to do with the direction of transcription.

Recently, analyses of promoters that apparently lack TATA elements have fueled further debate about the mechanisms of transcription initiation and the roles of TATA in determining the start site and direction of transcription. Studies of the SV40, TdT, DHFR, porphobilinogen deaminase, and ribosomal protein genes have revealed DNA sequence elements, called initiator elements, that overlap transcription start sites and direct start site placement (Ayer and Dynan, 1988; Smale and Baltimore, 1989; Means and Farnham, 1990; Hariharan and Perry, 1990; Beaupain et al., 1990). Transcription from the TdT element, termed an Inr (Inr will be used to refer to the TdT initiator and elements with homology to the TdT

1391

1392 Relative Activities of TATA and Initiator Elements

initiator; initiator elements without homology to the TdT element will not be referred to as Inrs, even though they may function by an identical mechanism), has been found to require TFIID for activity, even in the absence of a TATA box (Pugh and Tjian, 1990; Smale et al., 1990; Conaway et al., 1991). Furthermore, Inr elements are not restricted to promoters that lack TATA boxes, as an Inr also has been detected, both functionally and by sequence homology to the TdT Inr, at the transcription start site of at least one promoter that contains a TATA box, the AdML promoter (Hu and Manley, 1981; Smale et al., 1990; Conaway et al., 1990, 1991). In fact, long before analyses of promoters that lack TATA boxes, studies of several promoters that contain TATA boxes had demonstrated an important role for DNA sequences in the vicinity of the transcription start sites (Grosschedl and Birnstiel, 1980; Corden et al., 1980; Hu and Manley, 1981; Talkington and Leder, 1982; Dierks et al., 1983; Chen and Struhl, 1985). Grosschedl and Birnstiel (1980) first described a role for this “initiator” region in a histone H2A gene, which contains a TATA box.

A recent analysis by Carcamo et al. (1990) revealed homology to the TdT Inr at the start site of the Ad IVa2 gene, which had been thought for several years to lack a TATA box. Surprisingly, this study revealed the presence of a TATA box that, rather than being located upstream of the start site, was found downstream of the start site, between nucleotides +10 and +20. TFIID binds to this site and is required for IVa2 transcription. However, the most compelling evidence that TFIID actually functions by binding at this location is that a nucleotide substitution mutation, which converts the downstream TATA sequence (TATAGAAA) to a stronger consensus TATA sequence (TATAAA), increases activity of the promoter. Carcamo et al. (1990) proposed that in this promoter, the Inr element dominantly determines the transcription start site and the direction of transcription.

Based on the discoveries of initiator elements and of a potential downstream TATA element, it was necessary to re- evaluate the relative roles and contributions of TATA and Inr in localizing transcription start sites and in determining transcriptional directionality. To analyze start site placement, we tested the activity of synthetic promoters containing both TATA and Inr elements at various locations relative to each other. To analyze their contributions towards transcription directionality, we reversed the orientation of TATA either alone, in the presence of an upstream activator, or in the presence of Inr. Finally, we have searched for functional Inr activity in the start site regions of a variety of genes, which both contain and lack TATA elements. This analysis leads us to a model concerning the roles and mechanisms of action of TATA and Inr.

EXPERIMENTAL PROCEDURES AND RESULTS AND DISCUSSION^

Relative Activities of TATA and Inr in Localizing Transcrip- tion Start Sites and Communicating with Spl-We have shown previously that the Spl-dependent SV40 21-bp repeats can strongly activate transcription through either a TATA or an Inr element, which direct the activated transcription to a single start site (Smale et al., 1990). The TATA-mediated transcription began 25-30 bp downstream from TATA, and the Inr-mediated transcription began from within the Inr. Furthermore, when both TATA and Inr were placed down-

The “Experimental Procedures” are presented in the miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

stream from the Spl-dependent activator element, with the TATA box 25 bp from the Inr, transcription increased another 3-5-fold and initiated from sites within the Inr (Smale et al., 1990). In the experiments described here, we varied the locations of TATA and Inr relative to each other when downstream of the Spl-dependent SV40 21-bp repeats. These experiments were of interest for two reasons: 1) to address the relative strengths of the TATA and Inr elements in communicating with Spl molecules and in positioning transcription start sites, and 2) to determine if the Inr element could alter the strict requirement for a mammalian TATA box to be located 25-30 bp from the start site.

We first prepared plasmids containing synthetic promoters in which the distance between TATA and Inr was increased by 5, 10, or 15 bp from that found in the AdML promoter (where the distance between the final A of the TATAAA and the transcription start site is 25 bp). These supercoiled plasmids were then tested for promoter activity using in vitro transcription reactions with HeLa cell nuclear extracts. RNA transcribed in vitro was analyzed by primer extension analysis. Maximal transcription was observed with TATA positioned 25 bp from Inr (Fig. lA, lune 3) . This transcription was %fold more efficient than with either TATA or Inr alone (Fig. lA, lanes 1 and 2). When the distance between TATA and Inr was increased to 30, 35, or 40 bp, high levels of transcription began about 25 bp downstream of TATA (Fig. lA, lanes 4-6). As determined by densitometry analysis, 7- 12-fold lower levels of transcription began within the Inr (see long exposure in right panel of Fig. lA). Similar results were observed in four separate experiments using two different HeLa extract preparations. Moreover, although the experiments performed here involved 200 ng of template DNA and a 60-min reaction time, similar results were found in experiments with 800 ng of template and 20-min reaction times (data not shown).

We also decreased the distance between TATA and Inr in the vector containing the Spl sites. With a distance of 20 or 15 bp, almost all of the transcription again began 25 bp downstream of TATA, rather than from within the Inr (Fig. 2B, lunes 3-5). Transcription mediated by the Inr was more than 20-fold reduced from the transcription mediated by TATA. As described above, similar results were found with either 200 or 800 ng of DNA and with 20- or 60-min reaction times (data not shown).

The experiments in Fig. 1 were performed in the absence of a preincubation period with extract and DNA prior to the addition of nucleotide triphosphates. With this protocol, the results observed could be due to the fact that the formation of a preinitiation complex is more rapid with a TATA box than with an Inr. To address this possibility, we repeated the experiment in Fig. 1 with a protocol that included a 30-min preincubation period (at 30 “C) with extract and template prior to the addition of nucleotide triphosphates. The preincubation period was then followed by a 30-min reaction time. The results from this experiment (Fig. 2 A ) were similar to those described in Fig. 1.

Thus, in these competition experiments, TATA appeared to be more efficient than Inr in mediating activation by Spl and in localizing transcription start sites. This “dominance” of TATA relative to Inr may be a general feature of these elements or, alternatively, the element closest to the Spl sites may be dominant. More importantly, TATA and Inr appeared to act independently of each other, strongly suggesting that an Inr element is not capable of relaxing the strict spacing

Relative Activities of TATA and Initiator Elements 1393

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Spl sites TATUU Distance b.trre,n TATA and Inr

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VI -

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IX ,-e 35 bp

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Inr elements with variable spacing. In vitro transcription reactions were performed for 60 min with nuclear extracts derived from HeLa cells. RNA transcripts were analyzed by primer extension analysis with a SP6 promoter primer (Promega), followed by electro- phoresis of labeled cDNA products on an 8% denaturing polyacrylamide gel. A, transcription reactions contained 200 ng of plasmid V (Spl/TATA), VI (Spl/Inr), VI1 (Spl/TATA/Inr), VIII, IX, or X. Plasmids VI1 through X contained the Spl sites and both TATA and Inr, with the distance between the two elements of 25 (VII), 30 (VIII), 35 (IX), or 40 (X) bp. The arrow points to the location of bands derived from transcripts which initiated within the Inr in all plasmids (and also 25 bp downstream from TATA in plasmid VII). The bracket next to TATA-mediated points to the locations corresponding to transcripts which initiated about 25 bp downstream from TATA in plasmids VIII, IX, and X. Both short (left panel) and long (right panel) exposures of the polyacrylamide gel are shown. Similar results were found in four separate experiments using two different extract preparations. Some of the reactions were also repeated with two independent preparations of supercoiled template, yielding identical results. B, transcription reactions contained 200 ng of plasmids V, VI, VII, XI, or XII. Plasmids VII, XI, and XI1 contained the Spl sites and both TATA and Inr, with the distance between the two elements of 25 (VII), 20 (XI), or 15 (XII) bp. The arrow and brackets are as described for A. C, the plasmids used for the experiments in A and B are diagrammed schematically. Spl sites (black box) refer to the SV40 21-bp repeats, which contain five binding sites for transcription factor Spl (Dynan and Tjian, 1983). The directional AdML TATA element is indicated by the open triangle. The TdT Inr is indicated by the grey triangle. The arrows indicate the approximate locations of the transcription start sites found for each promoter. The plasmid number is indicated to the left and to the right is

1 2 3 4 5 6 7 - 8

T A W l n r (h. 2) +

1 2 3 4 - 5 6 1

FIG. 2. Activities of spacing mutants after preincubation with extract and in constructs lacking Spl-binding sites. A, in vitro transcription experiments were performed as described in the legend to Fig. 1, but with a preincubation step prior to beginning the reaction. The extract and template DNA were incubated at 30 "C for 30 min. Then, nucleotide triphosphates were added to begin the transcription reaction, which proceeded for an additional 30 min at 30 "C. RNA products were analyzed by primer extension analysis as above. No signal was observed if the reaction was carried out in the complete absence of nucleotide triphosphates (data not shown). B, in vitro transcription reactions were carried out with plasmids that vary the spacing between TATA and Inr, but unlike the plasmids in Figs. 1 and 2A, these plasmids lacked the Spl sites. Reactions were performed for 60 min with 800 ng of plasmid DNAs and 100 pg of HeLa extract. The plasmids used and distances between TATA and Inr are indicated above and below each lane. The reaction in lane 1 was performed with a template containing an isolated TATA box. The arrows and brackets indicate the locations or expected locations of the various primer extension products.

requirement between TATA and the transcription start site. Variation of Distance between TATA and Inr in the Absence

of Spl Sites-In Fig. 2B, the results are shown from an experiment similar to that described above, but with promoter constructs that lack Spl-binding sites. These results are important because they revealed an additional characteristic of TATA and Inr elements: an ability to cooperate with each other when separated by a distance of less than 25 bp. Fig. 2B, lane 1, shows the activity of a promoter containing an isolated TATA box. As is sometimes found with this very weak promoter, the specific signal is not detectable, although some nonspecific background bands are visible (see Smale et al., 1990). With both a TATA box and Inr at a distance of 25 bp, as in plasmid IV (Fig. 2B, lane 2), much higher levels of transcription were found (see also Fig. 5, lanes 15 and 16)) similar to the results reported previously (Smale et al., 1990). However, when the distance between TATA and Inr was increased to 30,35, or 40 bp, no specific initiation was detected

indicated the distance between TATA and Inr in plasmids that contain both (this distance refers to the distance between the final A of TATAAA and the A at the start site of the TdT Inr). Similar results were found in three separate experiments using two different extract preparations.


(Fig. 2B, lanes 3-5). Longer exposures of the gel from Fig. 2B revealed mostly background signals, suggesting that the TATA and Inr elements are directing extremely low levels of transcription, as is expected when the elements are acting independently of each other and of an upstream activator. Similar results were found in three separate experiments. This result shows that TATA and Inr cannot cooperate with each other when separated by 30 or more base pairs.

In contrast, when the distance between TATA and Inr was decreased to 20 or 15 bp (Fig. 2B, lanes 6 and 7), initiation was easily detected at the same locations as in Fig. lB, lanes 4 and 5. A similar result was found in three independent experiments. Therefore, these slightly heterogeneous start sites, which are located at a distance of approximately 25 bp downstream of the TATA box, are at much greater levels than the undetectable transcription directed by the TATA box in lanes 1,3,4, and 5. This result suggests that when the distance between TATA and Inr is reduced to 20 or 15 bp, the Inr facilitates transcription initiation. However, the location of the start site is determined predominantly by the TATA element. These results provide strong support for the idea that an Inr cannot influence the location of a transcription start site directed by a TATA box. Results described below (Fig. 9) confirm by an independent method that under special conditions, the Inr can indeed function even when transcription initiates slightly downstream from the normal +1 position.

Transcription from a Promoter Containing an Inr Upstream of a TATA Box-The experiments in Figs. 1 and 2 suggest either that TATA is the dominant element in communicating with the Spl sites or that the element closest to the Spl sites is dominant. To determine whether or not the element closest to the Spl sites is functionally dominant, we prepared a template in which a TATA box was located downstream from an Inr. In an in vitro transcription reaction, this construct also showed TATA dominance (Fig. 3, lane 3) . By densitometry, 10 times more transcription began 25 bp downstream from TATA than from within the upstream Inr. Moreover, the frequency of initiation from within the Inr was reduced by 10-fold from that found with a promoter lacking the downstream TATA oligonucleotide (Fig. 3, compare lanes 2 and 3) . Similar results were obtained in 11 separate experiments using 100,200,400, or 600 ng of template DNA. With 100 ng of template, the ratio between the two start sites found with plasmid pUSInr/TATA was reduced by a factor of 2 (data not shown). This result demonstrates that the preference of the transcription machinery for the TATA box is independent of whether TATA or Inr is closer to the Spl sites.

Transcription from Promoters with TATA and Inr Elements on Either Side of the Spl Sites-The experiments in Figs. 1- 3 demonstrate that TATA communicates with Spl more effectively than does Inr. This result inspired us to perform an additional experiment that provides an independent and more stringent test of the relative activities of TATA and Inr. This approach also allowed us for the first time to address the relative effectiveness of the TATA and Inr elements when functioning together. For these experiments, we placed one of the basal control elements (Inr or TATA) on one side of the Spl sites and then asked if transcription from this promoter could be influenced by placing a different basal element on the opposite side of the Spl sites. In this manner, we could determine if the transcription machinery, when given the choice between two core promoters located on different sides of the Spl sites, had a preference for a particular core control element. The results obtained with this approach, although

1 2 3 4

B Spl sites

V

pUSInr

pUSInr/TATA v FIG. 3. Activity of a promoter containing an Inr upstream

from TATA. A, in uitro transcription reactions were performed as described in the legend to Fig. 1, with 400 ng of template DNA. Plasmids used were V ( l a n e I, Spl/TATA), pUSInr ( l a n e 2, Spl/Inr), pUSInr/TATA (lane 3, containing TATA oligonucleotide inserted downstream of Inr in plasmid pUSInr), and pSP72 vector (lane 4 ) . The three arrows point to the cDNA products of interest. Next to the arrows are diagrams of the promoters, with the location of the transcription start sites (indicated by small arrows pointing to the right) for which the bands correspond. Similar results were observed in 11 separate experiments with three different extract preparations. Moreover, a similar ratio between TATA-mediated and Inr-mediated initiation was observed with pUSInr/TATA using either 200 or 600 ng of template, but the ratio was reduced by a factor of 2 when using 100 ng of template. B, the plasmids used in A are diagrammed schematically. The rectangles, triangles, and arrows are as described in the legend to Fig. 1. For plasmid pUSInr/TATA, the large arrow refers to the major start site and the small arrow refers to the minor start site.

much less dramatic than those in Figs. 1-3, are provided in Fig. 4.

First, we placed an Inr element on one side of the Spl sites (Fig. 4B, L side) and asked what effect the presence of TATA, Inr, or TATA + Inr would have on that transcription (in the L direction) if placed on the opposite side ( R side). We also confirmed the transcriptional activation in the opposite (R) direction with a different oligonucleotide primer (R primer). The purpose of this experiment was not to directly compare the levels of transcription in the R and L directions, as this type of comparison would be misleading because the Spl sites are not symmetrical. Instead, the purpose was to determine if the presence of a particular core element on the R side of the Spl sites could cause a reduction of Inr-mediated transcription in the L direction. Our results are shown in Fig. 4A. Transcription initiation from within the Inr was reduced only slightly by placing TATA or Inr on the opposite (R) side (lanes 2 and 3 ) , but by 4-fold with TATA + Inr on the R side (lane 4 ) . Transcription in the R direction was stimulated as expected (Fig. 4A, lanes 5-8). This 4-fold reduction was observed only with reactions performed with high amounts of template DNA (800 ng) and not with lower amounts (100 and 200 ng; data not shown). Furthermore, with variations of the reaction time or the amount of extract added to the reactions, we did not observe an effect of greater than 4-fold between the signals with plasmids LVI and LVI-RVII (data not


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FIG. 4. “Tug-of-war” experiment: activities of promoters containing Inr, TATA, or TATA + Inr on opposite sides of a Spl-dependent activator. A, in vitro transcription reactions were performed with 800 ng of template as described in the legend to Fig. 1. The plasmids used are listed above each lane and correspond to those diagrammed in B and described under “Experimental Proce- dures” and “Results.” The major band in each of lanes 1-16 corresponds to the band of interest. For lanes 17 and 18, the bracket indicates the heterogeneous bands of interest. Primer extension reactions were performed with the L (lanes 1-4,9-12, and 17-18) or R (lanes 5-8 and 13-16) primers. The specific activities of the L and R primers were comparable, with the L primer specific activity typically three times higher than the R primer activity. The panel for lanes 17 and 18 corresponds to a much longer exposure of the gel than do the other panels. The reductions observed in lanes 4 and 18 were observed with three different extract preparations, but only with the high amounts of template DNA used here and not with lower (100 or 200 ng) DNA concentrations (data not shown). The experiment in lanes 17 and 18 could not be performed a t lower DNA concentrations because under those conditions, the background signal was greater than the specific signal. For the particular experiments shown here, lanes I7 and 18 were performed with a different extract than were the remaining lanes. B, the plasmids used in A are diagrammed schematically. The rectangles, triangles, and arrows are as described in the legend to Fig. 1C. The plasmid names are indicated to the left, and the lanes in which the plasmid were tested are indicated to the right, as referring to whether the L or R primer was used for the reaction. The large arrows at the top refer to the regions where the L and R primers hybridize. For plasmids 111 and VII, the three arrows pointing to the left represent the 15 or so start sites detected in lane 17.

shown). Moreover, because the 21-bp repeats are not symmetrical, we performed the same experiments with the Spl sites in the opposite orientation and found a similar result, in that Inr-mediated transcription was reduced by TATA + Inr, while TATA had little effect (data not shown).

The above result suggests that when Spl is bound to the 21-bp repeats, it can facilitate transcriptional activation in either direction. The efficiency of initiation in each direction appears to result from the “preference” of the transcription machinery for the basal control elements ( i e . Inr, TATA, or

TATA + Inr) located on either side of the Spl molecules. In other words, the Spl molecules and general transcription machinery must choose between forming a preinitiation complex in the L direction or the R direction. In these experiments, the transcription apparatus apparently “prefers” to activate transcription through the TATA + Inr elements, rather than through just the Inr element, resulting in a 4-fold decrease in transcription in the direction of the Inr (L direction) when TATA + Inr are placed in the opposite (R) direction. However, the strong preference for TATA relative to Inr, described in Figs. 1-3, was not reflected in this experiment (Fig. 4A, lane 3).

Although a preference for TATA + Inr was found in Fig. 4A, lanes 1 4 , the reduction in Inr-mediated transcription in the L direction was small (only 4-fold). This small reduction may suggest that there is only a small preference for TATA + Inr relative to Inr. However, other issues must also be considered. For example, because the promoters contain 5 Spl-binding sites, it is possible that three Spl molecules can activate transcription in one direction as the other two simultaneously activate transcription in the opposite direction. In addition, it remains possible that each Spl site is capable of simultaneously activating transcription in both directions. These considerations may also explain why a preference was not found for TATA relative to Inr. However, despite these considerations, the effects observed in lanes 1-4, as well as in lanes 17 and 18 (see below) suggest that, at least to some degree, the transcription directed by the 5 Spl sites shows a preference for the TATA + Inr basal control elements.

In contrast to the effect on Inr-mediated transcription, TATA-mediated transcription was unaffected by placing TATA, Inr, or TATA + Inr in the opposite (R) direction (Fig. 4A, lanes 9-12). Thus, although Spl prefers to activate transcription through TATA + Inr instead of through Inr, it does not appear to prefer to activate transcription from TATA + Inr relative to TATA in this experiment. This conclusion is based on the observation that inclusion of TATA + Inr in the R direction does not reduce transcription from TATA in the L direction as it did from Inr in lune 4.

As a final test, we examined Spl-mediated transcription in the absence of TATA or Inr. With only the Spl-dependent 21-bp repeats, low levels of heterogeneous transcription were activated in both directions. Fig. 4A, lane 17, shows this transcription in the L direction (lanes 17 and 18 required a much longer autoradiographic exposure than did lanes 1-16). However, when TATA + Inr was added to the R side of the Spl sites, transcription in this direction was increased to the expected levels (not shown here, but see Fig. L4; plasmid VII; and Smale et dl., 1990), while transcription in the L direction was strongly reduced (Fig. 4A, lane 18). When either TATA or Inr was added to the R side, little or no reduction in L transcription was found (data not shown). These results demonstrate a stronger preference of the 21-bp repeats to activate transcription through a basal promoter consisting of TATA + Inr elements relative to a basal promoter containing neither element.

Although the experiments in Fig. 4 yielded results that were less dramatic than those presented in Figs. 1-3, they suggest that in at least one assay, the combination of TATA + Inr appears to be preferred by the transcription machinery. Taken together with the results from Figs. 1-3, these results may have implications about the mechanisms of transcriptional activation mediated by TATA and Inr. Possibly, the domi- nances and preferences that were observed suggest that the preinitiation complex directed by a TATA box is more stable or is formed more rapidly than that directed by an Inr.


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FIG. 5. Influence of an Inr on a directional TATA box. A, in uitro transcription reactions were performed as described in the legend to Fig. 1, using 400 (lanes 1-3 and 15-17), 600 (lanes 7-14), or 900 (lanes 4-6) ng of DNA. Quantitative comparisons cannot be made between the different panels (except for between lanes 11 and 12, which were spliced together from the same exposure of the same gel) because different DNA concentrations and primers were used. Such comparisons are not necessary for the conclusions drawn from these experiments. The DNA concentrations chosen for each panel were those that gave the clearest signal with the lowest level of background bands. The names of the plasmids tested are listed at the top and the primer used is shown at the bottom of each panel. The dots refer to the locations of the bands of interest for the adjacent lanes. Lanes 4-6 and 11-14 are from longer exposures (3 times) than are the other lanes. For the particular experiments shown here, the same extract was used for lanes 1-10 and a different extract for lanes 11-17. In multiple experiments and with various DNA concentrations, similar results were observed (with lower DNA concentrations the absolute signals were reduced, but the ratios between signals within each panel remained the same). Specifically, similar results to those in lanes 1-3 were observed in three separate experiments (using 100,200, or 400 ng of template), and were also observed with the Spl sites in the opposite orientation. For lanes 4-6, similar results were observed in six independent experiments with two different extract preparations, and with 200, 600, or 900 ng of template. For lanes 7- 9, similar results were observed in four experiments with two different extract preparations, and with 200, 600, or 900 ng of template. For lanes 11 and 12, similar results were found in two experiments. For lanes 13 and 14, similar results were found in three experiments with 200,600, and 900 ng of template. For lanes 15-1 7, similar results were found in four separate experiments with either 400 or 800 ng of DNA. R, at the top, the DNA sequences extending from just upstream of the TATA box to downstream of the transcription start sites (in the absence of an Inr) are indicated. The particular sequences are found in the plasmids listed to the left of each sequence. In bold-faced type are indicated the sequences that were reversed to produce the two

Similarly, the preinitiation complex directed by both TATA + Inr may be even more stable or formed more rapidly than with TATA alone. This differential formation rate or stability could result from either differential protein/DNA interactions or different mechanisms of interaction with Spl.

The experiments described in Fig. 4 also begin to address the determinants of transcriptional directionality from a promoter. The results suggest that TATA + Inr influences the direction of transcription not only by activating transcription in a particular direction, but also by causing a reduction in Spl-activated transcription in the opposite direction.

Relative Activities of TATA and Inr in Determining Tran- scriptional Directionality-As described in the Introduction, previous studies have suggested that TATA is a unidirectional element, which may impart directionality to transcription initiation as a result of its orientation within a promoter. However, the discoveries of Inr elements and of a potential downstream TATA box (Carcamo et al., 1990) suggest that an Inr element may be capable of dominantly determining the direction of transcription.

To begin to address the relative influences of TATA and Inr on the direction of transcription, we asked if an Inr in general could influence the unidirectional nature of TATA. In other words, can the presence of an Inr cause a TATA element to act in a completely bidirectional manner? More- over, can an Inr alter the direction of transcription from that dictated by the TATA box? We prepared promoter constructs containing a TATA element or one of two reverse orientation TATA elements in three different promoter environments: when downstream from the SV40 21-bp repeats, when present alone, and when 25 bp upstream from an Inr.

First, in the presence of the 21-bp repeats, both TATA (Fig. 5A, lane 1 ) and two reverse TATA constructs (Fig. 5A, lanes 2 and 3 ) directed transcription start sites to specific locations downstream of the Spl sites. Both reverse TATA elements, although positioned a t slightly different locations within the plasmid, directed transcription initiation from a similar distance of 24 nucleotides downstream. This result confirmed that the reverse TATA elements are functional and were responsible for localizing the transcription start site. How- ever, transcription initiation from both reverse TATA plasmids was reduced by ?"fold. We do not know whether the low level of transcription from these reverse TATA plasmids was a result of TFIID functioning as a partially bidirectional molecule, or whether a unidirectional TFIID binds to the template with low affinity in the opposite orientation. Re- gardless of the mechanism, the AdML TATA element clearly is capable of directing accurate transcription initiation when placed in either orientation downstream of the Spl sites. There is nothing intrinsic to this TATA element which pre- vents it from functioning in its reverse orientation, even though its level of activity was reduced.

When present alone, TATA directed extremely low levels of initiation in a crude nuclear extract (Fig. 5A, lane 4; this panel and the panel containing lanes 11-14 were from a three times longer autoradiographic exposure than were the remaining panels). Reversal of TATA resulted in a reduction of initiation frequency (lanes 5 and 6) ; we could not detect transcription, but the signal with TATA alone was very weak.

The results from Fig. 5, lanes 1-3 and 4-6, revealed that,

different reverse TATA contructs. The arrows indicate the location of the start sites directed by each TATA box, as determined with lanes 1 3 in A. At the bottom, the plasmid used for A is diagrammed schematically. The setup for this diagram is as described in the legends to Figs. 1 and 3. Arrows are indicated only for those transcripts that were looked for in the experiments in A. The dashed arrows indicate transcripts that were not detected.


as expected, the AdML TATA element is most highly active in its natural orientation, but retains some activity in its reverse orientation. This demonstration of the directional nature of TATA allowed us to perform the experiment of interest: to place an Inr element downstream of the normal and reverse TATA elements in order to determine if the presence of the Inr can cause the AdML TATA element to function equally well in either orientation. In the presence of Inr, we detected results similar to those found with TATA alone, in that reverse TATA appeared to be much less active than the normal TATA (Fig. 5A, lanes 7-10).

Thus, in this test, an Inr was not capable of significantly influencing the unidirectional nature of TATA. In other words, the presence of an Inr could not convert a reverse TATA element, which by itself only weakly activates transcription, into a stronger activator. This result suggests that the Inr does not influence the intrinsic preference of TFIID to bind TATA or activate transcription from TATA in a largely unidirectional manner.

One concern when manipulating the TATA element as in our experiments was that it might not function effectively in either orientation following reversal because of characteristics of the surrounding sequences. To address this issue, we first were careful to reverse, not only the TATAA, but also about 4 (rTATA-1) or 10 (rTATA-2) surrounding nucleotides. Moreover, we have tested experimentally transcription from two of these plasmids in the opposite direction. We found that following reversal, transcription in the opposite (L) direction proceeded at the level expected for a functional TATA element, both in the absence and presence of an Inr in that direction (Fig. 5A, lanes 11 and 12). In direct comparisons, these levels of transcription were about 3-fold higher than the levels detected with the R primer from plasmids I1 and IV (data not shown). This %fold difference was consistent with the 3-fold higher specific activity typically achieved with the labeled L primer. Thus, as expected, the reverse TATA elements apparently retained full TATA activity in the reverse orientation.

Because we detected transcription in the L direction from the reverse TATA construct, we asked if the presence of an Inr facing in the R direction could influence L transcription. This experiment was another test of the ability of the Inr to influence the direction of transcription as determined by TATA. It was possible that the presence of an Inr facing in the R direction might force the preinitiation complex to form over the TATA box in that direction, causing a reduction of the level of transcription directed by TATA in the L direction. However, we found that again the Inr had no influence on L transcription in this context (Fig. 5A, lanes 13 and 14).

Finally, we tested whether the Inr element was unidirectional or bidirectional. We found that a reverse orientation Inr element could not activate transcription directed from a TATA box (Fig. 5A, lanes 15-17). We also could not detect transcription in the opposite (L) direction with this promoter (data not shown).

These experiments failed to demonstrate a general effect of Inr on the directional nature of TATA. Although TATA was largely unidirectional, it clearly functioned in the reverse orientation when downstream of the 21-bp repeats (Fig. 5, lanes 1-3). The Inr did not allow TATA to act in a more bidirectional manner (Fig. 5, lanes 7-10) and did not influence the frequency of transcription initiation in the direction dictated by TATA (Fig. 5, lanes 13 and 14). Finally, the Inr element appeared inactive in its reverse orientation (lanes 15-17). These results suggest that, just as Inr cannot dominantly influence the localization of transcription start sites

when TATA is present (as in Figs. 1-3), it cannot influence the direction of transcription as determined by a TATA box. Although these experiments appear to contradict the previous study with the Ad IVa2 promoter (Carcamo et al. 1990; see Introduction), the Ad IVa2 experiments involved distances between the downstream TATA and Inr (10-20 bp) that were not tested here. At those distances, Inr may indeed influence TATA and TFIID function.

The Orientation of TATA within a Promoter Might Not Determine the Direction of Transcription-Although the above data confirmed that a consensus TATA box is more active in one orientation than in the other, they caused us to re-evaluate the data suggesting that the orientation of TATA is the sole determinant of the direction of transcription from a promoter. Clearly, this strong consensus TATA element had significant activity in its reverse orientation. This observation is consistent with the recent demonstration that some TATA elements are symmetrical and that TFIID interacts functionally with a wide range of AT-rich sequences (Hahn et al., 1989), making it unlikely that all are completely unidirectional. Instead, our data with these synthetic promoters are equally consistent with alternative models: a simple alternative is that, within a unidirectional promoter, transcriptional directionality might be determined not by the orienta- tions of TATA and Inr, but instead by the location of these elements relative to an upstream activator. A second simple alternative is that transcription from promoters containing the AdML TATA box always proceeds in both directions, with seven times more transcription (on the basis of the quantitation in Fig. 5, lanes 1-3) occurring in one direction than in the other.

To begin to distinguish between these models, we tested the transcriptional activities in both directions from plasmid L/rVII, shown in Fig. 6. In this plasmid, we placed the 21-bp repeats downstream of the TATA and Inr elements (with TATA 25 bp from Inr; Fig. 6B, plasmid L/rVII). We then tested for transcription in both directions. This promoter directed efficient transcription from 25 bp downstream of TATA, but in the L direction away from the Inr (Fig. 6A, lane 5) . This transcription was at the same level as from a promoter containing a reverse TATA element downstream from the Spl sites, in the absence of Inr (Fig. 6A, lane 4 ) . Interest- ingly, we were unable to detect transcription in the opposite (R) direction, using either the R primer (Fig. 6A, lane 7) or a primer complementary to the 21-bp repeats (data not shown). Thus, the direction of transcription from this promoter appears to be determined by the relative locations of the upstream activator and TATA within the promoter. In this case, the TATA element functions as reverse TATA and, consistent with Fig. 5 (lane 17), the Inr is nonfunctional, simply because of their locations relative to the upstream activator.

We have not eliminated the possibility that the direction of transcription from this promoter would be in the R direction if the Spl sites were at a different distance downstream from the transcription start site, or if binding sites for an activator other than Spl were used. Possibly, our results concerning the determinants of transcriptional directionality are unique for Spl, and results with other activators would be different. Further experiments are needed before we can draw conclusions about the role of TATA in determining the direction of transcription from a promoter. However, our results clearly demonstrate that the determinants of directionality from any promoter have not yet been defined and that several alternative models are equally likely. Although some natural promoters contain TATA elements that do not retain activity when reversed (Nagawa and Fink, 1985; Bielin-


1 2 3 4 5 1 6 7

u u Primer L R

B L I” 2- s I aitea _R

W I I TATAAPI .

w- f .* ?

UrV

Urvll 7 , . +?

FIG. 6. Direction of transcription from promoters containing an “apparently downstream” activator element. A, in vitro transcription reactions were carried out as described in the legend to Fig. 1, with 200 ng of template DNAs. The plasmid names are indicated at the top and the primer used, at the bottom. The major bands in lanes 1-5 correspond to the bands of interest. No major cDNA products were detected in lunes 6 and 7. Similar results were found in three separate experiments with 200 or 800 ng of template DNA. B, the plasmids used in A are diagrammed schematically. The objects and labeling are as described in the legends to Figs. 1 and 4. The dashed arrows pointing to the right, followed by question marks, indicate that we cannot detect transcription in this direction and we do not know what is necessary for transcription to proceed in this direction.

ska et al., 1989), the orientation of this element may have very little to do with the direction in which transcription proceeds. It was not determined if reversal of those TATA elements activated transcription in the opposite direction.

Our results suggest that the determinant of directionality in a simple synthetic promoter may be the location of the TATA and/or Inr elements relative to the activator. In natural and more complex promoters, this simple mechanism may also determine transcriptional directionality. However, this model does not account for the action of activators located downstream of the transcription start site. Possibly, higher order structural characteristics of a promoter are important for directionality. For example, an activator like Spl may function from a downstream position only if an appropriate activator is positioned upstream from the TATA box. Another possibility is that downstream activators are fundamentally different from upstream activators. This argument is consistent with recent studies of the Drosophila GAGA factor (Cros- ton et aL, 1991), which functions from either an upstream or a downstream location. Experiments have suggested that this activator does not communicate directly with the general transcription machinery, but activates transcription solely by facilitating the removal of histone H1 or nucleosomes from the transcription start site region (Croston et al., 1991). This role could be carried out equally well with the element either upstream or downstream of the start site.

Prevalence of Transcriptional Initiator Elements-TATA elements are found in the promoters of many, if not most, genes transcribed by RNA polymerase 11. Homology to the TdT Inr has been found at the transcription start sites of some promoters that either contain or lack TATA elements (e.g. the AdML, Ad IVa2, sea urchin histone (Grosschedl and

Birstiel, 1980), osteonectin (McVey et al., 1988), and interleu- kin 6 (Ray et al., 1990) promoters). In addition, initiator elements that have little or no homology to the TdT Inr have been described in other protein-coding genes (the SV40 late (Ayer and Dynan, 1988), DHFR (Means and Farnham, 1990), and ribosomal protein (Harihan and Perry, 1990) genes). Still other promoters display characteristics suggesting that, although they contain little sequence homology to the TdT Inr, they may contain an element with functional initiator activity. The B-cell specific 1B9 gene (Hermanson et al., 1988) appears to lack a TATA element, but begins transcription from a single nucleotide? In addition, the Ad E1B and human immunodeficiency virus type 1 promoters contain TATA boxes, but begin transcription from the correct nucleotide after the TATA box has been eliminated (Bielinska et al., 1989; Wu and Berk, 1988). To address the prevalence of initiator-like elements and the possible heterogeneity of DNA sequences that can impart initiator function, we tested and compared the start site regions from some of these genes.

The start site regions were inserted into plasmid 111, which contains the SV40 21-bp repeats with no TATA or Inr (Smale et al., 1990). All start site regions were inserted such that transcription from the authentic initiation sites would produce the same size transcript, labeled as +1 in Fig. 7 , A and B. As shown previously (Smale et al., 1990), plasmid I11 does not itself contain a functional TATA element between the 21-bp repeats and the inserted Inr. More recent experiments have confirmed these results by demonstrating that TFIID cannot bind to this region:

Two characteristics of the transcription from these plasmids were analyzed in order to determine whether or not initiator-like activity was present. First, we looked for the abilities of the start site regions to focus the transcription to a specific location that corresponds to the start site in vivo for the endogenous gene. Second, we analyzed the abilities of the start site regions to stimulate Spl-activated transcription to levels that are above background.

Fig. 7A (left panel) shows the activities of several start site regions when using 100 ng of template DNA. Similar results were found with 200 or 400 ng of template (data not shown). The right panel (Fig. 7 A ) shows 100 ng of the identical samples, but in transcription reactions with 100 ng of an internal control plasmid containing the SV40 21-bp repeats upstream of a TATA element. Lane 1 is the negative control, showing the transcriptional activity from a promoter containing the Spl sites in the absence of an Inr. Lane 2 shows the activity of a promoter containing the TdT Inr downstream of the 21-bp repeats, and lane 3, the activity of a similar promoter containing a previously described Inr mutant, which alters nucleotides +3 to +5 and results in a strong reduction in Inr activity (Smale et al., 1990). Both the strength of the promoter and the accuracy of initiation are affected by this mutation, although about 50% of the transcription still begins at the correct start site. Lanes 4 and 5 show the activities of the Inr elements found at the start sites for the AdML and Ad IVa2 genes, both of which exhibit homology to the TdT Inr (Fig. 7B) . Both of these regions contain functional Inr activity, although reduced from the levels found with the TdT Inr. The activities of the Ad E1B and 1B9 start site regions (see above and Fig. 7 B ) are shown in Fig. 7A, lanes 6 and 7. Both of these regions exhibit initiator-like activities, in that they focus transcription initiation to the authentic start site and stimulate transcription to levels above background. These start site regions appear to be about as active as the AdML

‘ W. Wood and R. Wall, personal communication. F. Pugh and R. Tjian, personal communication.

Relative Activities of TATA and Initiutor Elements 1399 A

I- +I - C’

r ~ . a t r m c t 3 20 3 20 3 20 3 20

+I !L Inr c c c T c A T T c T G G A G A cl 100s Inr Yutant [C C C C T C A TIC 0 A [ G G A G A C1 6 MYL W T [ C C T C A l C m C T T C C G 25 Ad IV.2 C G T k T U ] G A C m T C C G 26 Ad CIB T T 0 C m T m C A T C T m C C T 18

189 A TDT A E X I G A G A A A c A C ~ 30 RIV-I dhf L

G T A m G G G m C T C T 0 G tl

SVlO )(L @ A C G I T J T L ~ ~ T C A m C C C A 4 c om30 c l r a l r AWTEJC G G 56

FIG. 7. Prevalence of initiator elements. A, in vitro transcription reactions were carried out as described in the legend to Fig. 1, but with only 100 ng of template DNA. The start site regions from various genes were inserted into the Sac1 and BarnHI sites of a plasmid containing the SV40 21-bp repeats with no TATA element (see “Results” and “Experimental Procedures”). All inserts were such that if transcription began at the authentic in oioo start site for the particular gene, it would be at the position corresponding to the arrow labeled +1. Lane 1 shows the activity of the plasmid containing no start site region insertion. Lane 2 shows the activity of the TdT Inr, and lane 3 the activity of the F3 Inr mutant described previously (Smale et al., 1990). The start site regions used for l anes 4-10 are indicated above the lane. The left panel shows the activities of the plasmids when tested alone. This experiment is representative of three separate experiments, using two different extract preparartions. Similar results were also found with 200 or 400 ng of template DNA, with the absolute signals from each template increasing by similar degrees with increasing DNA concentration. The right p a n e l shows the activities of the plasmids (100 ng) in reactions containing an equal amount (100 ng) of an internal control (plasmid V), with the S p l sites and TATA (labeled C). We do not know whether the doublets observed in some lanes at the correct start site (e .g l anes 3, 6, and 7) indicate two distinct start sites or whether they are artifacts of incomplete primer extension reactions. B, the DNA sequences of the various start site regions are shown. The in oiuo transcription start site for each is labeled as +l. The nucleotides in boxes correspond to those that are homologous to the same region in the T d T Inr.

and Ad IVa2 Inrs, even though they contain no homology to the TdT Inr. The human immunodeficiency virus type 1 start site region contains some homology to the TdT Inr, but as described previously (Smale et al., 1990), does not activate transcription to levels above background. The initiator element for the DHFR gene was previously described by Means and Farnham (1990) and functions effectively as described ( l a n e 9) . The SV40 major late start site region, although important for SV40 transcription (Ayer and Dynan, 1988), exhibits only a low level of initiator activity (Fig. 7A, lane 10). Moreover, the initiation sites observed with the SV40 start site region ( l a n e 10) did not correspond to that found in the authentic promoter (Ayer and Dynan, 1988).

The experiment in Fig. 7 revealed the activities of various start site regions when placed downstream from binding sites for Spl, in the absence of a TATA box. A second characteristic of the TdT Inr is that it can strongly facilitate transcription directed by a TATA box in the absence of an upstream activator (see, for example, Fig. 5, lanes 15 and 16). Experi- ments have shown that with low amounts of extract, the Inr only weakly activates TATA-mediated transcription relative to a promoter containing an isolated TATA box (see also Fig.

1 2 3 4 5 6 7 8

FIG. 8. Activities of various initiator elements placed downstream of the AdML TATA box. The start site regions indicated in Fig. 7 R for the 1R9, Ad EIB, Ad IVa2, and TdT genes were placed in plasmid 11, downstream of the AdML TATA box. I n uitro transcription reactions were then performed with 600 ng of these planmids in addition to an equimolar amount of plasmid 11, containing a promoter with an isolated TATA box. Reactions contained either 3 or 20 pl of HeLa extract (30 or 200 pg) . Arrows point to the location8 of the cDNA producb resulting from transcription either of the promoter containing both the TATA box and start site region, or of the promoter containing only the TATA box (plasmid 11). Similar results were found in three separate experiments.

8, lane 7).6 In contrast, with higher amounts of extract (Fig. 8, lane 8) , the TATA/Inr transcription increases, but the TATA transcription decreases. This decrease in TATA transcription may result from the inactivation of the weak TATA promoter by nonspecific DNA-binding proteins like histone H1 (Croston et af., 1990). The presence of the Inr appears to prevent the promoter from being inactivated.

As an independent method for testing the activities of various start site regions, we placed some of them at the appropriate distance downstream from the AdML TATA box. We then tested the activities of these plasmids with low and high amounts of extract. The results are shown in Fig. 8. The TdT Inr (lanes 7 and 8) facilitated TATA transcription by 4- fold when tested with 30 pg of extract (3 pl, compare upper and lower bands in lane 7). With 200 pg of extract (20 pl, lane 8) the difference between TATA/Inr and TATA is greater than 20-fold. With the 1B9 (lanes 1 and 2). Ad E1B (lanes 3 and 4 ) , and Ad IVa2 (lanes 5 and 6) start site regions, the results were consistent with those found in Fig. 7. All three start site regions had little or no influence on TATA transcription when tested with low amounts of extract. However, with high extract, all three start site regions to some degree prevent the template from the inactivation that apparently is the result of nonspecific DNA-binding proteins. The magni- tudes of the signals and the degrees of resistance to inactivation are similar to the results in Fig. 7.

Based on these results, the TdT Inr appears to be the most effective initiator that we have tested. However, we cannot make quantitative comparisons because our constructs contain only the sequences between nucleotides -6 and +11 in each gene. Some of the better characterized genes appear to require a larger region for optimal initiator activity or may be augmented by adjacent sequence elements (Means and Farn- ham, 1990; Garfinkel et al., 1990; Carcamo et af., 1990). In addition, the initiators for the E1B and 1B9 genes have not been analyzed in enough detail to determine if a larger region might be required for optimal initiator activity.

The most important conclusion from this experiment is that many of the start site regions contain clear initiator

“Zenzie-Gregory, B., O’Shea-Greenfield, A., and Smale, S. T. (1992) J. Biol. Chern. 267, in press.


activity, but no initiator consensus sequence can be defined. Thus, several different classes of initiator elements may exist, each of which is recognized by a distinct sequence-specific DNA-binding protein. Alternatively, these data may suggest that initiator elements function by a mechanism that does not involve a high affinity or high specificity protein/DNA interaction (see below).

Initiator Actiuity Does Not Depend on Transcription Zniti- ation at an Adenosine Residue-The only homology evident between the various active initiator elements in Fig. 7 was the adenosine at nucleotide +l. Our previous mutagenesis had shown that, in the TdT Inr, this A was critical for activity (Smale et al., 1990). Fig. 9 shows that transcription does not need to begin at this A for the Inr to be active. I t was shown previously that the start site for Ad EIV transcription could be altered by varying the concentrations of individual nucleotide triphosphates (suggesting that the K,,, for the first nucleotide in the RNA transcript is higher than the K,,, for the nucleotides required for elongation; Samuels et al., 1984). Thus, if we lower the concentration of ATP, such that transcription cannot begin efficiently a t an adenosine, we might expect either a reduction in the efficiency of initiation or a shift of the transcription start site. As shown in Fig. 9, we found the latter result. Omission of ATP from the transcription reaction did not affect the rate of transcription initiation (low levels of ATP were apparently present in the dialyzed extract), but shifted the transcription start site downstream from the adenosine residue, to the cytosine and/or thymidine residues (Fig. 9, lane 2). Increasing amounts of ATP restored the majority of initiation to the correct nucleotide (lanes 3- 6) . Titration of AMP-PNP, which contains a nonhydolyzable P-7 bond, also restored transcription to the correct nucleotide (lanes 9-12). This control showed that the effect observed did not result from depletion of the substrate needed for B-7 ATP hydrolysis, which is required for RNA polymerase I1 initiation (Sawadogo and Roeder, 1984). Furthermore, omission of GTP or CTP from the transcription reaction had little influence on the start site or initiation frequency (lanes 14 and 15).

NTP

1 2 3 4 5 6 7 8 9 10 11 12 1314 1 5 1 6 ~ ".,. _i.

+1+ *+

A.1 * * . - I+ I+*

C C C T C A T T C T G G A G A FIG. 9. Inr-mediated transcription can begin at nucleotides

other than adenosine. In oitro transcription reactions were performed with 800 ng of plasmid LVI (Spl/Inr) as described in the legend to Fig. 1. Lanes I , 7 and 13 show reactions with crude nuclear extracts to which 250 PM ATP, UTP, CTP, and GTP had been added. For lanes 2 and 7, ATP was not added to the reactions, but the extracts apparently contain low levels of ATP. For lanes 3-6, increasing amounts of ATP were added. In lanes 9-12, increasing amounts of AMP-PNP were added. Similar results were found in three separate experiments. As controls, GTP (lane 1 4 ) , CTP (lane 15), or U T P (lane 16) were omitted from the reactions. The + I arrow indicates initiation at the A, and the arrow with the asterisk indicates downstream initiation sites. At the bottom is shown the TdT Inr sequence, with the +1 and downstream start sites indicated.

Omission of UTP strongly reduced transcription (lane 16), most likely because of a lower concentration of residual UTP within the dialyzed extract. Taken together, these results demonstrate that although the functional Inr focuses transcription to the adenosine, it is just as active in stimulating Spl-directed transcription from a different nucleotide if necessary.

By What Mechanism Does an Initiator Element Function?- The purpose of this study was to address the relative roles of TATA and Inr in determining, for a simple promoter, the direction of RNA polymerase I1 transcription as well as the location of the transcription start site. However, the results found have implications concerning the mechanism by which an Inr functions.

The demonstration that several initiator-like elements do not have significant sequence homologies with each other might suggest that several classes of initiators exist, each of which is recognized by a distinct binding protein. Indeed, a protein has been identified in crude nuclear extracts that binds with high affinity to the DHFR initiator element (Means and Farnham, 19901, but no protein appears to strongly interact with the TdT Inr.fi Possibly the DHFR, TdT, 1B9, and E1B elements are representative of four distinct initiator classes that will interact with four distinct proteins.

However, one alternative is that all of these elements function in the same manner. They might interact with a known component of the general transcription machinery, which recognizes all of the various sequence elements with a low affinity and low specificity. This component could be RNA polymerase 11, TFIID, a TFIID-associated protein, or TFIIB (reviewed in Sawadogo and Sentenac, 1990).

A second alternative is that these initiator elements may function without being recognized by a sequence-specific DNA-binding protein. An Inr might stimulate transcription by promoting the formation of a melted or open structure that is then weakly recognized by the general transcription machinery. Several different DNA sequence elements might be able to form such a structure, explaining the lack of a consensus element. Although Inr elements are not AT-rich and potential hairpin structures are not evident, little is known about alternate DNA structures. Inconsistent with this model, however, is our result demonstrating that the TdT Inr is unidirectional. It might be expected that if the sole function of an Inr were to promote template melting, it would be functional in either orientation.

What Role Does an Znr Play within a Promoter?-Our data suggest that an Inr has no dominance over the activities of a TATA box. What, then, is the primary role of an Inr within a promoter? To attempt to answer this question, considera- tion must be given to the fact that Inr elements are found in promoters that either contain or lack TATA boxes. In promoters lacking a TATA element, the Inr is responsible for directing RNA polymerase I1 to a precise transcription start site. I t is not clear if it is important for transcription from some TATA-less genes to begin a t a specific nucleotide, while transcription from others begins a t multiple start sites sometimes spanning several hundred nucleotides. Possibly, precise initiation is important for appropriate RNA processing, stability, or transport of some genes. Alternatively, precise initiation may be important for a promoter to incorporate the activities of the multiple positive and negative regulatory elements that surround the start site. However, unlike the T d T gene, many genes regulated during lymphocyte-differ- entiation do not contain TATA or Inr element, and begin

fi S. Smale, unpublished observations.


transcription from multiple sites (Lo et al., 1991). Some of these genes contain promoters with considerable homology to the TdT promoter, except for the Inr. Thus, the ability of an Inr to localize transcription to a single start site might not be its primary role.

A second role for an Inr in a simple promoter without a TATA box is that it influences the direction of transcription. An Spl-dependent upstream activator directs low levels of transcription from heterogeneous start sites in both directions. Insertion of an Inr on one side strongly stimulates transcription only in that direction. However, natural promoters are thought to be more complex than the simple promoters used in this study. The direction of transcription in those cases might be determined by the relative locations of other activator elements within the promoter. Furthermore, TATA and initiator elements are not likely to be the sole determinants of directionality because many promoters (e.g. those from which transcription initiates at several different start sites) do not appear to contain either. Thus, it is not clear if the ability of an Inr to influence the direction of transcription is important to natural promoters, even those that lack TATA boxes.

In addition to considering the role of an Inr in genes that lack TATA boxes, it may be more informative to consider the role of an Inr in genes that also contain a TATA box (e.g. AdML promoter). In these promoters, Inr does not appear to be important for determining the location of the start site or the direction of transcription (except possibly in specialized cases (Carcamo et al., 1990)). Instead, its primary activity, if properly located relative to the TATA box, is to increase promoter strength. If bound by a high affinity DNA-binding protein, the Inr may act like an upstream activator and may play a role in regulation of transcription. However, if it is recognized by a component of the general transcription machinery or if it functions solely through structural considerations, an Inr would serve as a means of modulating basal promoter strength from gene to gene. Just as some TATA elements (e.g. TATAAAA) appear to facilitate higher levels of transcription than others, the presence of an Inr downstream of TATA may also lead to a stronger basal promoter. In this regard, the role of an Inr may be similar to the roles of the region between TATA and Inr in the AdML promoter (Conaway et al., 1990,1991) and of the region downstream of the start site in the gfa promoter (Nakatani et al., 1990a, 1990b). Both of these regions increase the strength of basal promoters and are thought to interact with TFIID or TFIID- associated proteins. Therefore, in all of these cases, modula- tion of promoter strength might require no proteins other than the general transcription apparatus, with the various elements increasing the formation rate or stablity of the preinitiation complex. Finally, this same role of modulating basal promoter strength may also be the primary role of an initiator in promoters that lack TATA boxes, with secondary

roles being influences on the direction of transcription and the location of the start site.

Acknowledgments-We are grateful to Arnold Berk, Michael Carey, Albert Courey, and Randy Wall for critical reading of the manuscript, and to Frank Pugh, Robert Tjian, Will Wood, and Randy Wall for communicating results prior to their publication.

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1402 Relative Activities of TATA and lnitiator Elements

roles of tata and initiator elements in determining the start site

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