Orientation of the transcription preinitiation complex in Archaea Stephen D. Bell*, Peter L. Kosa†‡, Paul B. Sigler†§, and Stephen P. Jackson*¶ *The Wellcome Trust and Cancer Research Campaign Institute of Cancer and Developmental Biology, Tennis Court Road, Cambridge, CB2 1QR, United Kingdom; and †Department of Molecular Biophysics and Biochemistry and the §Howard Hughes Medical Institute, Yale University, 260 Whitney Avenue͞JWG423, P.O. Box 208114, New Haven, CT 06520-8114 Contributed by Paul B. Sigler, October 5, 1999 The basal transcription machinery of Archaea corresponds to the binding activities (e.g., see refs. 14 and 15), it is likely that minimal subset of factors required for RNA polymerase II transcrip- TAF–DNA interactions may influence the orientation of TFIID tion in eukaryotes. Using just two factors, Archaea recruit the RNA on some promoters. polymerase to promoters and define the direction of transcription. Although the above models may account for orientational Notably, the principal determinant for the orientation of transcrip- specificity in TBP–TATA-box recognition in eukarya, they are tion is not the recognition of the TATA box by the TATA-box- unlikely to do so in archaea. aTBP is a considerably more binding protein. Instead, transcriptional polarity is governed by the symmetric molecule than its eukaryal homologues: the two interaction of the archaeal TFIIB homologue with a conserved motif repeats in eTBP have 28–30% identity, whereas in aTBP they are immediately upstream of the TATA box. This interaction yields an Ϸ40% identical. Crucially, the charge potential distribution in archaeal preinitiation complex with the same orientation as the aTBP is highly symmetric (16) and, furthermore, the proline analogous eukaryal complex. clash model (12) cannot apply to aTBP because the analogous positions in both repeats are occupied by proline. Finally, both he basal transcription machinery of Archaea is strikingly biochemical and genome sequence analyses suggest that Archaea Tsimilar to the core components of the eukaryal RNA poly- do not possess TAFs (6, 17, 18), implying that TAFs arose after merase (RNAP) II apparatus (1, 2). Archaeal promoters contain the separation of archaeal and eukaryal lineages. It is likely, a TATA box found 25 bp upstream of the transcription start site therefore, that the evolutionary progenitor of the eukaryal and that forms a ternary complex with the archaeal TATA-box- archaeal transcription machineries relied on factor(s) other than binding protein (aTBP) and the archaeal homologue of tran- TBP to define transcriptional polarity. scription factor TFIIB, TFB (3, 4). It is possible to reconstitute It is therefore possible that TFB and͞or RNAP plays impor- transcription from a range of archaeal promoters by using tant roles in determining the orientation of transcription in recombinant aTBP, TFB, and highly purified RNAP; no addi- Archaea. In eukarya, TFIIB can influence the orientation of tional transcription factors are required even on highly positively binding of eTBP to the TATA box (13), and this property of supercoiled templates (5–7). The crystal structure of the ternary TFIIB might be attributable to the recently described interaction complex of aTBP and the C-terminal core domain of TFB between TFIIB and the BRE sequence element found upstream (TFBc) on DNA is highly similar to the corresponding structure of the TATA box in some strong promoters (19). An analogous in eukaryotes. However, the orientation of aTBP and TFBc with motif has been identified in the strong archaeal T6 promoter of respect to the transcription start site was inverted in the archaeal the Sulfolobus shibatae virus SSV1 (20). In the current work, we complex compared with the homologous eukaryal TBP͞TFIIB͞ demonstrate that the archaeal BRE is found in a range of TATA-box structure (8, 9). promoters of various strengths and that the TFB–BRE interac- The formation of the aTBP͞TFB͞TATA-box ternary complex tion is the principal element which defines transcription polarity. is required for recruitment of the RNAP to the transcription We also demonstrate that the absolute polarity of the archaeal initiation site, downstream of the TATA box. Clearly, it is of key ternary complex is the same as the homologous eukaryal TBP– importance that transcription occurs in a unidirectional manner TFIIB–DNA complex. at most promoters and, thus, a mechanism must exist to ensure that RNAP is recruited in a directional manner. The simplest Materials and Methods model to explain the directional recruitment of RNAP would be Plasmid Construction. Plasmid pINIT was generated by annealing to define the polarity of the preinitiation complex at the first the self-complementary oligonucleotide INIT and ligating into step; the binding of TBP to the TATA box. However, despite BamHI-digested pBluescript. Insertion of the INIT oligonucle- extensive analysis in both Eucarya and Archaea, whether and otides removed the BamHI sites flanking the insert. Subsequent how TBP binds the TATA element in a preferred orientation and TATA-box-containing oligonucleotides were inserted into the how transcriptional polarity is established are still poorly under- internal BamHI site in pINIT. stood. Recent studies suggest that the intrinsic deformability of the TATA box may be important, indicating that the C-terminal Oligonucleotides. Name is followed by sequence; X ϭ 5-bromode- repeat of eukaryal TBP (eTBP) preferentially contacts the more oxyuridine. flexible end of the TATA box (10). However, the molecular basis INIT, GATCTTGAACCCTCTATCGGATCCGATAGAG- of this preference is unclear. The highly asymmetric charge GGTTCAA; potential distribution in eTBP may also play a role (11). An alternative model suggests that the asymmetric positioning of a proline in the C-terminal repeat may contribute to orientational Abbreviations: RNAP, RNA polymerase; TBP, TATA-box-binding protein; aTBP and eTBP, archaeal and eukaryal TBPs; TFBc, C-terminal core domain of TFB; TAF, TBP-associated specificity (12). However, experiments using eTBP derivatized factor; BRE, TFB-responsive element; EMSA, electrophoretic mobility-shift assay. with a DNA scission reagent have shown that eTBP has only a ‡Present address: Harvard University School of Medicine, Department of Biological Chem- modest (60:40) preference for binding the TATA box of the istry and Molecular Pharmacology, Boston, MA 02115. adenovirus major late promoter and yeast CYC1 promoters in a ¶To whom reprint requests should be addressed. E-mail: [email protected]. particular orientation (13). In eukaryal nuclei, eTBP is com- The publication costs of this article were defrayed in part by page charge payment. This plexed with associated factors, TAFs. As a number of TAFs in article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. the TFIID complex have been demonstrated to possess DNA- §1734 solely to indicate this fact. 13662–13667 ͉ PNAS ͉ November 23, 1999 ͉ vol. 96 ͉ no. 24 Downloaded by guest on September 23, 2021 EF1-T, GATCCATGTGCGAAAGCTTTAAAAAGTAA- UV-Mediated Photocrosslinking. Binding reactions were set up as GTTCAAAAGT; described above with 5 fmol (Ϸ20,000 cpm) of probe and 1 pmol EF1-L, GATCACTTTTGAACTTACTTTTTAAAGCTT- of TBP and͞or TFB as indicated. Reactions were incubated for TCGCACATAG; 10 min at 48°C before irradiation at 310 nm for 18 min with a UV TATAINV-T, GATCCTATGTGCGAAAGCTTTTTAAA- transilluminator (Photodyne, New Berlin, WI). Samples were GTAAGTTCAAAAGT; boiled in SDS͞PAGE loading buffer before electrophoresis on TATAINV-L, GATCACTTTTGAACTTACTTTAAAAA- an SDS͞12% polyacrylamide gel. GCTTTCGCACATAG; FLIP2.3-T, GATCCTATGTGCGAATACTTTAAAAAGC- Results TAGTTCAAAAGT; To identify the sequences and factors responsible for determin- FLIP2.3-L, GATCACTTTTGAACTAGCTTTTTAAAGT- ing the orientation of transcription from archaeal promoters, we ATTCGCACATAG; used the TATA-box region from the promoter for the Pyrococcus FLIP2.6-T, GATCCTATGTGCACTTACTTTAAAAAGC- woesei ef1␣ gene, previously used in structural studies (9). We TTTCTCAAAAGT; first tested whether the initiation site of transcription has a role FLIP2.6-L, GATCACTTTTGAGAAAGCTTTTTAAAGT- in determining transcriptional polarity. Thus, we generated AAGTGCACATAG; pINIT, the plasmid construct shown in Fig. 1a, in which two FLIP2.10-T, GATCCTATTTGAACTTACTTTAAAAAG- copies of an 18-nucleotide region encompassing the character- CTTTCGCACAAGT; ized initiation site of the Sulfolobus shibatae virus (SSV1) T6 FLIP2.10-L, GATCACTTGTGCGAAAGCTTTTTAAAG- gene were present in inverted repeat configuration flanking a TAAGTTCAAATAG; BamHI restriction site. This BamHI site was then used to create UPINV-T, GATCCTATGTGCGAAAGCTTTAAAAAGC- pEF1 by inserting 36 nucleotide residues corresponding to the TTTCTCAAAAGT; ef1␣ gene TATA box and flanking regions, such that the UPINV-L, GATCACTTTTGAGAAAGCTTTTTAAAGC- midpoint of the TATA box was equidistant from the upstream TTTCGCACATAG; and downstream initiation sites. RNAs arising from in vitro DOINV-T, ATCCTATGTGCACTTACTTTAAAAAGTA- transcription of these plasmids in either orientation can be AGTTCAAAAGT; detected by primer extension, using either T3 or T7 sequencing DOINV-L, GATCACTTTTGAACTTACTTTTTAAAGT- primers (see Fig. 1a). In vitro transcription assays, using recom- AAGTGCACATAG; binant aTBP and TFB and highly purified RNAP, were per- T6WT-T, GATCTAGATAGAGTAAAGTTTAAATACTT- formed with the parental pINIT construct and with the construct ATATAGATAGA; (pEF1) containing wild-type ef1␣ sequences inserted