Proc. Nati. Acad. Sci. USA Vol. 88, pp. 8052-8056, September 1991 Biochemistry
The initiator directs the assembly of a transcription factor IID-dependent transcription complex JUAN CARCAMO, LEONARD BUCKBINDER*,
AND
DANNY REINBERGt
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854-5635
Communicated by Keith R. Yamamoto, June 10, 1991 (received for review February 26, 1991)
ABSTRACT Highly purified RNA polymerase H was found to be able to weakly recognize the initiator (Inr) present in the adenovirus IVa2 and major late promoters. The association of RNA polymerase H with the Inr was enhanced by the general transcription factors. The Inr was capable of directing the formation of a DNA-protein complex. Transcription competent complexes on the adenovirus major late and IVa2 promoters appear to be formed by alternative pathways mediated through the Inr and/or "TATA" motif. The presence of both motifs, however, is required for efficient transcription utilizing a discrete start site. Complexes formed at either site required transcription factor TFIID, the TATA binding protein. Consistent with this observation, a TFUD requirement was demonstrated for transcription from a mutant adenovirus major late promoter construct lacking a functional TATA motif.
promoters were oriented in opposite directions. Accordingly, a model was presented in which the polar nature of the initiator governed the direction of transcription (16).
MATERIALS AND METHODS DNA Constructs. Plasmid DNA containing the wild-type and mutated Ad-IVa2 promoters were as described (16). Insertion mutants between the Ad-IVa2 Inr and TATA motifs were generated using oligonucleotide-directed mutagenesis using the Amersham mutagenesis kit. The sequence of the various insertion mutants are indicated in Fig. 2A. The sequences of the various Ad-MLP constructs are shown in Fig. 1C. Mutation in the Ad-MLP TATA motif(deletion and inversion) in the context of the wild-type promoter were constructed as follows. A recombinant M13 phage [AC40 (17)] containing AdMLP sequence from positions -260 to + 190 was used in the construction of the TATA-null and TATA inversion mutants. A Xba 1-HindIII fragment was isolated and cloned into a chloramphenicol acetyltransferase expression vector derived from pSV2-CAT. Protein Factors and Transcription Reactions in Vitro. Transcription conditions were as described (16). Products of the reactions were analyzed by primer-extension as described (16). RNA polymerase II was purified to apparent homogeneity (see Fig. IA; ref. 26). The protein preparation was devoid of contaminating general transcription factors. Transcription factors TFIIA (14), -IIB (11), -IIE (18), and -IIF (19) were purified as described. TFIID was purified to homogeneity as described from recombinant Escherichia coli cells expressing the yeast TFIID gene (11).
Studies on various class II promoters have indicated that distinct cis-acting DNA elements can affect transcription (1, 2). One of these elements is the "TATA box," which in higher eukaryotes is located -30 nucleotides upstream from the initiation site and is thought to position the start site of transcription (3, 4). The initiator (Inr) constitutes a second such element that appears to be present in most RNA polymerase II transcribed genes and encompasses the transcriptional start site (5). Specific initiation of transcription from class II promoters requires at least six protein transcription factors (TFIIA, -IIB, -IID, -IIE, -IIF, and -IIH) in addition to RNA polymerase II (6, 7). TFIID is the only general transcription factor containing DNA binding activity specific for the TATA motif (8, 9). Studies have demonstrated that binding of TFIID to the TATA motif is the first step in the formation of a transcription-competent complex, providing a target site for association of the other general transcription factors and RNA polymerase II (6, 10-14). Although it was thought initially that most of the RNA polymerase II-transcribed genes contained a TATA motif, it is now clear that many genes do not (15). We began our studies on TATA-less promoters by analyzing the factors required for transcription of the adenovirus IVa2 promoter (Ad-IVa2). The transcriptional start site of the Ad-IVa2 promoter is located 210 nucleotides upstream of the cap site of the adenovirus major late promoter (Ad-MLP) (15). Transcription from the Ad-IVa2 and ML promoters occurs on different DNA strands (15). Using recombinant yeast TFIID, we demonstrated that transcription of this so called TATAless promoter was dependent on TFIID (16). Upon inspection of IVa2 promoter sequences, a TATA-like sequence (5'TATAGAAA-3') was discovered -20 nucleotides downstream of the IVa2-transcriptional start site (16). An initiator motif was identified in the Ad-IVa2 promoter; in contrast to the TATA motifs, the initiator motifs in the IVa2 and ML
RESULTS The Inr Is Recognized by RNA Polymerase H. To analyze whether RNA polymerase II could recognize the Inr, a highly purified preparation of the enzyme (Fig. LA) was incubated under transcription conditions with a plasmid DNA containing either the wild-type (Fig. 1B, lanes 1-6) or a mutated (lanes 7-12) IVa2 Inr. Sites recognized by RNA polymerase II and, therefore, serving as transcriptional start sites were mapped using a primer-extension assay. In agreement with previous experiments, multiple transcriptional start sites were observed (20); however, some preferred sites were mapped to the Inr and the downstream TATA motif (Fig. 1B, lanes 1-3), and transcription from these sites was sensitive to a-amanitin (2 ,Lg/ml, data not shown). Those transcripts that initiated at position +1 required a wild-type Inr, as mutations of the conserved nucleotides in this element abolished position +1 initiation by purified RNA polymerase II (Fig. 1B, Abbreviations: Inr, initiator; ML, major late; MLP, ML promoter; Ad, adenovirus. *Present address: Department of Embryology, Carnegie Institute of Washington, Baltimore, MD 21210. tTo whom reprint requests should be addressed at: Department of Biochemistry, University of Medicine and Dentistry of New Jersey, 675 Hoes Lane, Piscataway, NJ 08854-5635.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
8052
Biochemistry: Carcamo et al.
Proc. Natl. Acad. Sci. USA 88 (1991)
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FIG. 1. Purified RNA polymerase II can recognize the Inr present in the Ad-IVa2 and ML promoters. (A) Silver staining of an SDS/polyacrylamide gel containing purified RNA polymerase II (hPol II) used in the experiments described in B and C. (B) Transcription reaction mixtures containing various amounts of RNA polymerase II (lanes 1-3, 7-9, and 13-15) or purified general transcription factors (lanes 4 and 10), or both purified RNA polymerase II and the general transcription factors (GTF) (lanes 5 and 11) were incubated with various IVa2 promoter constructs (4 ,ug/ml), as indicated (+, added; -, not added). Lanes 6 and 12 contain products of reactions performed with HeLa cell nuclear extract. Position +1 initiation and other initiation sites are indicated on the side. Schematic representation of the IVa2 constructs are shown under the autoradiograph. The boxes indicate the TATA and Iur motifs. The boldface type in the boxes indicates the mutations (16). (C) Reactions were as described in B, but the plasmid DNA used in the analysis contained the wild-type Ad-MLP (lanes 1-5) or an Ad-MLP with mutations in the Inr (lanes 6-9) as indicated under the autoradiograph.
lanes 7-9). When a wild-type IVa2 promoter served as the template, the addition of the general transcription factors to transcription reaction mixtures containing purified RNA polymerase II had two effects: transcription levels starting at position +1 were stimulated (compare lane 2 with 5) and transcription from sites other than position +1 was drastically reduced; thus specific initiation of transcription was restored (Fig. 1B, lane 5). Addition of the general transcription factors to reaction mixtures containing an IVa2 promoter with a mutated Inr abolished transcription completely (Fig. 1B, lane 11). Similar results were observed when sequences from the Ad-MLP extending from positions -38 to +10 were used in place of the Ad-IVa2 promoter (Fig. 1C). However, although initiation at position + 1 by purified RNA polymerase II was absolutely dependent on the Inr (Fig. 1C, compare lanes 1 and 2 with lanes 6 and 7), addition of the general factors resulted in some transcription from the Inr-mutated construct that started at and around position +1 (Fig. 1C, lane 9). The ability of the Ad-MLP to initiate transcription (in the presence of the general transcription factors) despite a mutated Inr might be explained by its TATA motif that could direct the formation of a transcriptionally competent complex. In the context of the Ad-MLP, mutations in the Inr are tolerated, whereas in the IVa2, such mutations abolish transcription. Mutations in the downstream IVa2-TATA motif that resulted in a drastic decrease of the levels of specific initiation and accuracy of position + 1 initiation (see below) did not abolish initiation by purified RNA polymerase II within this
site or at position +1 (Fig. 1B, lanes 13-15). These results demonstrate that purified RNA polymerase II can weakly recognize the Inr and that the general transcription factors confer specificity to RNA polymerase II for sequences within the Mr. The Ad-IVa2 mr Includes the Transcriptional Start Site and Functions Independently of the TATA Motif. Previous studies have indicated that the TATA motif present in the Ad-MLP regulates levels of transcription and accuracy of position +1 initiation (3, 21). To analyze whether the downstream TATA and/or the Inr motifs fix the initiation of transcription in the Ad-IVa2 promoter, these two DNA elements were separated by insertion of 5, 10, and 15 nucleotides. The resulting plasmid DNAs (Fig. 2A) were analyzed for transcription using a HeLa cell nuclear extract and the start sites were mapped by a primer-extension assay. The three insertion mutants (Inser-5, Inser-10, and Inser-15) retained position + 1 initiation, as the primer-extended molecules increased in size proportional to the nucleotides inserted (Fig. 2B, lanes 4-6, 7-9, and 10-12, respectively). The insertion mutant constructs also started transcription at sites other than position +1. These same cryptic initiation sites were also utilized when the TATA motif was mutated (Fig. 2B, lanes 13-15) but were absent in the wild-type construct (Fig. 2B, lanes 1-3). Transcription from these sites was sensitive to a-amanitin (2 ,ug/ml, data not shown). The levels of position + 1 initiation observed from the insertion mutants were similar to those observed from the TATA mutant construct (Fig. 2B). The levels of transcription from the Ad-MLP, added as an internal
Proc. Natl. Acad. Sci. USA 88 (1991)
Biochemistry: Carcamo et al.
8054
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control to the various reaction mixtures, were not affected (Fig. 2B, Lower). These results demonstrate that the start site of transcription in the Ad-IVa2 promoter is governed by and located within the Inr and that the spacing between the Inr and the downstream TATA motif affects levels of transcription. These results are in agreement with the studies of Baltimore and coworkers (5, 22) analyzing the Inr present in the terminal deoxynucleotidyltransferase and ML promoters and strongly suggest that the Inr can direct the formation of a transcriptionally competent complex and that the TATA and Inr act cooperatively to enhance transcription levels and to improve the specificity of the start site. The large decrease in transcription observed with the insertion mutants and the presence of multiple start sites indicates that the downstream TATA motif in the IVa2 promoter has a major role in directing levels of transcription and accuracy of position +1 initiation. The TATA Motif Present in the Ad-MLP Can Be Deleted Without Affecting Position +1 Initiation. Previous studies have indicated, using a run-off assay, that the Ad-MLP TATA motif is required for specific initiation of transcription in vitro (3, 17, 18, 21, 23). In light of the results presented above, we reanalyzed whether position +1 initiation and/or
the levels of transcription were affected by removing or inverting the TATA motifpresent in the Ad-MLP. In contrast to studies analyzing transcription from the galactose 1 promoter in yeast (24), inversion of the Ad-MLP TATA motif had no effect on position + 1 initiation in vitro (Fig. 3A, lanes 4-6) or in vivo (Fig. 3D, lane 2) but the levels of transcription were reduced. Binding ofTFIID to the inverted TATA motifs was observed and the footprint was similar to that observed with the wild-type construct (Fig. 3B). These results could be explained as follows: (i) the inversion of the TATA motif resulted in the creation of another TATA-like motif that was recognized by TFIID and/or (ii) TFIID may be a symmetrical molecule (25) or may function as a dimer. Either case, however, possess a problem: how does a symmetrical molecule or a TFIID molecule that is unable to distinguish the orientation ofits recognition site direct position +1 initiation? A likely explanation is that TFIID could, directly or indirectly, recognize DNA elements other than the TATA motif. Thus, we analyzed the effect of replacing the Ad-MLP TATA motif by random sequences (5'-TGACGGT-3', null). As expected, binding of TFIID was not observed with the null construct (Fig. 3B). Although the levels of transcription from the null mutant were drastically reduced in vivo using a transient expression assay (Fig. 3D, compare lanes 3 and 4) and in vitro using a nuclear extract (Fig. 3A, compare lanes 7-9 with lanes 1-3), position +1 initiation was retained; however, other minor sites surrounding position + 1 were also used as transcriptional start sites. These results indicate that the TATA motif helps to fix precisely the transcriptional start site and affects the levels of transcription. To analyze whether TFIID was required for transcription of the null construct, a system reconstituted with highly purified general transcription factors was used. Transcription was not detected in the absence of TFIID (Fig. 3C, lane 2); however, the addition of human TFIID (lane 3) or recombinant yeast TFIID (lanes 4 and 5) restored transcription. Initiation was heterogeneous starting at and around position + 1 and the levels of transcription were lower as compared to the wild-type Ad-MLP template (compare lanes 3-5 with lane 1). The results presented above strongly suggest that the TATA binding protein is required for transcription regardless of the presence of the TATA motif and, therefore, that TFIID does not necessarily function through a TATA motif but may be drawn into the transcription cycle by other elements (proteins and/or DNA sequences). Similar conclusions were obtained by Smale et al. (22) analyzing the Inr present in the terminal deoxynucleotidyltransferase and ML promoters. Thus, on TATA-less promoters, it is likely that TFIID enters the transcription cycle by interacting with other transcription factors and/or RNA polymerase II. The Inr Motif Directs the Formation of a TFHD-Dependent DNA-Protein Complex. Using the gel-mobility-shift assay, this laboratory demonstrated (ref. 11; J.C., 0. Flores, and D.R., unpublished data) that the entry of RNA polymerase II into the transcription cycle was dependent on a DNA-protein complex formed at the TATA motif and surrounding sequences of the Ad-MLP and included TFIIA, -IID, and -IIB (DAB complex). In addition, the binding of RNA polymerase II to the DAB complex was dependent on TFIIF (12). When a 74-base-pair DNA fragment containing the Ad-IVa2 Inr (positions -16 to + 10) and vector sequences was used in the DNA binding assay, the characteristic DA and DAB complexes were not observed (Fig. 4A, lanes 1-3). However, a DNA-protein complex was observed in the presence of highly purified TFIIA, -IIB, -1ID, and -IIF and RNA polymerase II (Fig. 4A, lane 5). The formation ofthis complex was dependent on TFIIF (lane 4), TFIID (lane 6), TFIIB (lane 8), and RNA polymerase II (lane 9) but independent of TFIIA (lane 7). The formation of the DNA-protein complex on this TATA-less DNA fragment was specifically competed by a
Biochemistry: Carcamo et al.
Proc. Natl. Acad. Sci. USA 88 (1991)
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The initiator directs the assembly of a transcription factor IID-dependent transcription complex JUAN CARCAMO, LEONARD BUCKBINDER*,
AND
DANNY REINBERGt
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854-5635
Communicated by Keith R. Yamamoto, June 10, 1991 (received for review February 26, 1991)
ABSTRACT Highly purified RNA polymerase H was found to be able to weakly recognize the initiator (Inr) present in the adenovirus IVa2 and major late promoters. The association of RNA polymerase H with the Inr was enhanced by the general transcription factors. The Inr was capable of directing the formation of a DNA-protein complex. Transcription competent complexes on the adenovirus major late and IVa2 promoters appear to be formed by alternative pathways mediated through the Inr and/or "TATA" motif. The presence of both motifs, however, is required for efficient transcription utilizing a discrete start site. Complexes formed at either site required transcription factor TFIID, the TATA binding protein. Consistent with this observation, a TFUD requirement was demonstrated for transcription from a mutant adenovirus major late promoter construct lacking a functional TATA motif.
promoters were oriented in opposite directions. Accordingly, a model was presented in which the polar nature of the initiator governed the direction of transcription (16).
MATERIALS AND METHODS DNA Constructs. Plasmid DNA containing the wild-type and mutated Ad-IVa2 promoters were as described (16). Insertion mutants between the Ad-IVa2 Inr and TATA motifs were generated using oligonucleotide-directed mutagenesis using the Amersham mutagenesis kit. The sequence of the various insertion mutants are indicated in Fig. 2A. The sequences of the various Ad-MLP constructs are shown in Fig. 1C. Mutation in the Ad-MLP TATA motif(deletion and inversion) in the context of the wild-type promoter were constructed as follows. A recombinant M13 phage [AC40 (17)] containing AdMLP sequence from positions -260 to + 190 was used in the construction of the TATA-null and TATA inversion mutants. A Xba 1-HindIII fragment was isolated and cloned into a chloramphenicol acetyltransferase expression vector derived from pSV2-CAT. Protein Factors and Transcription Reactions in Vitro. Transcription conditions were as described (16). Products of the reactions were analyzed by primer-extension as described (16). RNA polymerase II was purified to apparent homogeneity (see Fig. IA; ref. 26). The protein preparation was devoid of contaminating general transcription factors. Transcription factors TFIIA (14), -IIB (11), -IIE (18), and -IIF (19) were purified as described. TFIID was purified to homogeneity as described from recombinant Escherichia coli cells expressing the yeast TFIID gene (11).
Studies on various class II promoters have indicated that distinct cis-acting DNA elements can affect transcription (1, 2). One of these elements is the "TATA box," which in higher eukaryotes is located -30 nucleotides upstream from the initiation site and is thought to position the start site of transcription (3, 4). The initiator (Inr) constitutes a second such element that appears to be present in most RNA polymerase II transcribed genes and encompasses the transcriptional start site (5). Specific initiation of transcription from class II promoters requires at least six protein transcription factors (TFIIA, -IIB, -IID, -IIE, -IIF, and -IIH) in addition to RNA polymerase II (6, 7). TFIID is the only general transcription factor containing DNA binding activity specific for the TATA motif (8, 9). Studies have demonstrated that binding of TFIID to the TATA motif is the first step in the formation of a transcription-competent complex, providing a target site for association of the other general transcription factors and RNA polymerase II (6, 10-14). Although it was thought initially that most of the RNA polymerase II-transcribed genes contained a TATA motif, it is now clear that many genes do not (15). We began our studies on TATA-less promoters by analyzing the factors required for transcription of the adenovirus IVa2 promoter (Ad-IVa2). The transcriptional start site of the Ad-IVa2 promoter is located 210 nucleotides upstream of the cap site of the adenovirus major late promoter (Ad-MLP) (15). Transcription from the Ad-IVa2 and ML promoters occurs on different DNA strands (15). Using recombinant yeast TFIID, we demonstrated that transcription of this so called TATAless promoter was dependent on TFIID (16). Upon inspection of IVa2 promoter sequences, a TATA-like sequence (5'TATAGAAA-3') was discovered -20 nucleotides downstream of the IVa2-transcriptional start site (16). An initiator motif was identified in the Ad-IVa2 promoter; in contrast to the TATA motifs, the initiator motifs in the IVa2 and ML
RESULTS The Inr Is Recognized by RNA Polymerase H. To analyze whether RNA polymerase II could recognize the Inr, a highly purified preparation of the enzyme (Fig. LA) was incubated under transcription conditions with a plasmid DNA containing either the wild-type (Fig. 1B, lanes 1-6) or a mutated (lanes 7-12) IVa2 Inr. Sites recognized by RNA polymerase II and, therefore, serving as transcriptional start sites were mapped using a primer-extension assay. In agreement with previous experiments, multiple transcriptional start sites were observed (20); however, some preferred sites were mapped to the Inr and the downstream TATA motif (Fig. 1B, lanes 1-3), and transcription from these sites was sensitive to a-amanitin (2 ,Lg/ml, data not shown). Those transcripts that initiated at position +1 required a wild-type Inr, as mutations of the conserved nucleotides in this element abolished position +1 initiation by purified RNA polymerase II (Fig. 1B, Abbreviations: Inr, initiator; ML, major late; MLP, ML promoter; Ad, adenovirus. *Present address: Department of Embryology, Carnegie Institute of Washington, Baltimore, MD 21210. tTo whom reprint requests should be addressed at: Department of Biochemistry, University of Medicine and Dentistry of New Jersey, 675 Hoes Lane, Piscataway, NJ 08854-5635.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
8052
Biochemistry: Carcamo et al.
Proc. Natl. Acad. Sci. USA 88 (1991)
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FIG. 1. Purified RNA polymerase II can recognize the Inr present in the Ad-IVa2 and ML promoters. (A) Silver staining of an SDS/polyacrylamide gel containing purified RNA polymerase II (hPol II) used in the experiments described in B and C. (B) Transcription reaction mixtures containing various amounts of RNA polymerase II (lanes 1-3, 7-9, and 13-15) or purified general transcription factors (lanes 4 and 10), or both purified RNA polymerase II and the general transcription factors (GTF) (lanes 5 and 11) were incubated with various IVa2 promoter constructs (4 ,ug/ml), as indicated (+, added; -, not added). Lanes 6 and 12 contain products of reactions performed with HeLa cell nuclear extract. Position +1 initiation and other initiation sites are indicated on the side. Schematic representation of the IVa2 constructs are shown under the autoradiograph. The boxes indicate the TATA and Iur motifs. The boldface type in the boxes indicates the mutations (16). (C) Reactions were as described in B, but the plasmid DNA used in the analysis contained the wild-type Ad-MLP (lanes 1-5) or an Ad-MLP with mutations in the Inr (lanes 6-9) as indicated under the autoradiograph.
lanes 7-9). When a wild-type IVa2 promoter served as the template, the addition of the general transcription factors to transcription reaction mixtures containing purified RNA polymerase II had two effects: transcription levels starting at position +1 were stimulated (compare lane 2 with 5) and transcription from sites other than position +1 was drastically reduced; thus specific initiation of transcription was restored (Fig. 1B, lane 5). Addition of the general transcription factors to reaction mixtures containing an IVa2 promoter with a mutated Inr abolished transcription completely (Fig. 1B, lane 11). Similar results were observed when sequences from the Ad-MLP extending from positions -38 to +10 were used in place of the Ad-IVa2 promoter (Fig. 1C). However, although initiation at position + 1 by purified RNA polymerase II was absolutely dependent on the Inr (Fig. 1C, compare lanes 1 and 2 with lanes 6 and 7), addition of the general factors resulted in some transcription from the Inr-mutated construct that started at and around position +1 (Fig. 1C, lane 9). The ability of the Ad-MLP to initiate transcription (in the presence of the general transcription factors) despite a mutated Inr might be explained by its TATA motif that could direct the formation of a transcriptionally competent complex. In the context of the Ad-MLP, mutations in the Inr are tolerated, whereas in the IVa2, such mutations abolish transcription. Mutations in the downstream IVa2-TATA motif that resulted in a drastic decrease of the levels of specific initiation and accuracy of position + 1 initiation (see below) did not abolish initiation by purified RNA polymerase II within this
site or at position +1 (Fig. 1B, lanes 13-15). These results demonstrate that purified RNA polymerase II can weakly recognize the Inr and that the general transcription factors confer specificity to RNA polymerase II for sequences within the Mr. The Ad-IVa2 mr Includes the Transcriptional Start Site and Functions Independently of the TATA Motif. Previous studies have indicated that the TATA motif present in the Ad-MLP regulates levels of transcription and accuracy of position +1 initiation (3, 21). To analyze whether the downstream TATA and/or the Inr motifs fix the initiation of transcription in the Ad-IVa2 promoter, these two DNA elements were separated by insertion of 5, 10, and 15 nucleotides. The resulting plasmid DNAs (Fig. 2A) were analyzed for transcription using a HeLa cell nuclear extract and the start sites were mapped by a primer-extension assay. The three insertion mutants (Inser-5, Inser-10, and Inser-15) retained position + 1 initiation, as the primer-extended molecules increased in size proportional to the nucleotides inserted (Fig. 2B, lanes 4-6, 7-9, and 10-12, respectively). The insertion mutant constructs also started transcription at sites other than position +1. These same cryptic initiation sites were also utilized when the TATA motif was mutated (Fig. 2B, lanes 13-15) but were absent in the wild-type construct (Fig. 2B, lanes 1-3). Transcription from these sites was sensitive to a-amanitin (2 ,ug/ml, data not shown). The levels of position + 1 initiation observed from the insertion mutants were similar to those observed from the TATA mutant construct (Fig. 2B). The levels of transcription from the Ad-MLP, added as an internal
Proc. Natl. Acad. Sci. USA 88 (1991)
Biochemistry: Carcamo et al.
8054
5'-CTCG|GAGACCT*lCTCGGACCACTCTGAGACGAAGGC-3'
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FIG. 2. Transcription template activity of IVa2 promoter constructs bearing insertion mutations between the downstream TATA box and Inr motif. (A) Schematic representation of the IVa2 promoter constructs bearing various insertions between the TATA and Inr motifs. Dots indicate the nucleotides inserted. Underlined sequences represent the Inr element. Boxes indicate the downstream TATA motif. The construct labeled IVa2-TATA-mut contains substitutions in the downstream TATA motif as indicated. (B) Transcriptional template activity of the constructs shown in A. Reaction conditions were as described and contained HeLa cell nuclear extracts and various amounts of the constructs shown in A, as indicated above the lanes. Reaction mixtures also contained, as an internal control, a plasmid DNA (2 gg/ml) with the Ad-MLP [pD-139 DNA (13)]. Reaction products were analyzed by primer extension as described (16).
control to the various reaction mixtures, were not affected (Fig. 2B, Lower). These results demonstrate that the start site of transcription in the Ad-IVa2 promoter is governed by and located within the Inr and that the spacing between the Inr and the downstream TATA motif affects levels of transcription. These results are in agreement with the studies of Baltimore and coworkers (5, 22) analyzing the Inr present in the terminal deoxynucleotidyltransferase and ML promoters and strongly suggest that the Inr can direct the formation of a transcriptionally competent complex and that the TATA and Inr act cooperatively to enhance transcription levels and to improve the specificity of the start site. The large decrease in transcription observed with the insertion mutants and the presence of multiple start sites indicates that the downstream TATA motif in the IVa2 promoter has a major role in directing levels of transcription and accuracy of position +1 initiation. The TATA Motif Present in the Ad-MLP Can Be Deleted Without Affecting Position +1 Initiation. Previous studies have indicated, using a run-off assay, that the Ad-MLP TATA motif is required for specific initiation of transcription in vitro (3, 17, 18, 21, 23). In light of the results presented above, we reanalyzed whether position +1 initiation and/or
the levels of transcription were affected by removing or inverting the TATA motifpresent in the Ad-MLP. In contrast to studies analyzing transcription from the galactose 1 promoter in yeast (24), inversion of the Ad-MLP TATA motif had no effect on position + 1 initiation in vitro (Fig. 3A, lanes 4-6) or in vivo (Fig. 3D, lane 2) but the levels of transcription were reduced. Binding ofTFIID to the inverted TATA motifs was observed and the footprint was similar to that observed with the wild-type construct (Fig. 3B). These results could be explained as follows: (i) the inversion of the TATA motif resulted in the creation of another TATA-like motif that was recognized by TFIID and/or (ii) TFIID may be a symmetrical molecule (25) or may function as a dimer. Either case, however, possess a problem: how does a symmetrical molecule or a TFIID molecule that is unable to distinguish the orientation ofits recognition site direct position +1 initiation? A likely explanation is that TFIID could, directly or indirectly, recognize DNA elements other than the TATA motif. Thus, we analyzed the effect of replacing the Ad-MLP TATA motif by random sequences (5'-TGACGGT-3', null). As expected, binding of TFIID was not observed with the null construct (Fig. 3B). Although the levels of transcription from the null mutant were drastically reduced in vivo using a transient expression assay (Fig. 3D, compare lanes 3 and 4) and in vitro using a nuclear extract (Fig. 3A, compare lanes 7-9 with lanes 1-3), position +1 initiation was retained; however, other minor sites surrounding position + 1 were also used as transcriptional start sites. These results indicate that the TATA motif helps to fix precisely the transcriptional start site and affects the levels of transcription. To analyze whether TFIID was required for transcription of the null construct, a system reconstituted with highly purified general transcription factors was used. Transcription was not detected in the absence of TFIID (Fig. 3C, lane 2); however, the addition of human TFIID (lane 3) or recombinant yeast TFIID (lanes 4 and 5) restored transcription. Initiation was heterogeneous starting at and around position + 1 and the levels of transcription were lower as compared to the wild-type Ad-MLP template (compare lanes 3-5 with lane 1). The results presented above strongly suggest that the TATA binding protein is required for transcription regardless of the presence of the TATA motif and, therefore, that TFIID does not necessarily function through a TATA motif but may be drawn into the transcription cycle by other elements (proteins and/or DNA sequences). Similar conclusions were obtained by Smale et al. (22) analyzing the Inr present in the terminal deoxynucleotidyltransferase and ML promoters. Thus, on TATA-less promoters, it is likely that TFIID enters the transcription cycle by interacting with other transcription factors and/or RNA polymerase II. The Inr Motif Directs the Formation of a TFHD-Dependent DNA-Protein Complex. Using the gel-mobility-shift assay, this laboratory demonstrated (ref. 11; J.C., 0. Flores, and D.R., unpublished data) that the entry of RNA polymerase II into the transcription cycle was dependent on a DNA-protein complex formed at the TATA motif and surrounding sequences of the Ad-MLP and included TFIIA, -IID, and -IIB (DAB complex). In addition, the binding of RNA polymerase II to the DAB complex was dependent on TFIIF (12). When a 74-base-pair DNA fragment containing the Ad-IVa2 Inr (positions -16 to + 10) and vector sequences was used in the DNA binding assay, the characteristic DA and DAB complexes were not observed (Fig. 4A, lanes 1-3). However, a DNA-protein complex was observed in the presence of highly purified TFIIA, -IIB, -1ID, and -IIF and RNA polymerase II (Fig. 4A, lane 5). The formation ofthis complex was dependent on TFIIF (lane 4), TFIID (lane 6), TFIIB (lane 8), and RNA polymerase II (lane 9) but independent of TFIIA (lane 7). The formation of the DNA-protein complex on this TATA-less DNA fragment was specifically competed by a
Biochemistry: Carcamo et al.
Proc. Natl. Acad. Sci. USA 88 (1991)
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