Orientation of the transcription preinitiation complex in archaea

Abstract

The basal transcription machinery of Archaea corresponds to the minimal subset of factors required for RNA polymerase II transcription in eukaryotes. Using just two factors, Archaea recruit the RNA polymerase to promoters and define the direction of transcription. Notably, the principal determinant for the orientation of transcription is not the recognition of the TATA box by the TATA-box-binding protein. Instead, transcriptional polarity is governed by the interaction of the archaeal TFIIB homologue with a conserved motif immediately upstream of the TATA box. This interaction yields an archaeal preinitiation complex with the same orientation as the analogous eukaryal complex.

Figure 1

Sequences flanking the TATA box are important for defining the orientation of transcription. (a) Diagram of plasmid constructs used in transcription assays. pINIT was created by ligating oligonucleotides corresponding to an inverted repeat of the T6 transcription initiation region into pBluescript. Subsequently, other oligonucleotides corresponding to derivatives of the Pyrococcus woesei ef1α promoter were inserted into the BamHI site of pINIT. The sequence of these inserts is shown. Sequences upstream of the TATA box in the wild-type promoter are shown in reverse shading; sequences naturally occurring downstream of the TATA box are boxed; and the TATA box itself is contained within an arrow. The T3 and T7 sequencing primer annealing sites are shown by open wide triangles and the T6 start sites are indicated by filled arrows and labeled A and B. The open arrows indicate the unexpected additional start sites arising at the junction of the cloned sequences and the parental polylinker that are recognized by the RNA polymerase alone and are totally independent of TBP and TFB. (b) The ef1α TATA box and flanking sequences direct unidirectional transcription. pINIT and pEF1 were used in in vitro transcription assays and transcripts detected by using primer extension with either T3 or T7 sequencing primer, as indicated. The transcript initiating at the downstream T6 start site is indicated with a solid arrowhead. The second, factor-independent, start site is indicated by an open arrowhead. The identities of the start sites were confirmed by electrophoresis adjacent to dideoxynucleotide DNA sequence ladders prepared with the radiolabeled T3 and T7 primers. (c) Sequences flanking the TATA box govern orientation of transcription. The various plasmid constructs shown in a were used in in vitro transcription assays. Annotation is as above.

Figure 2

The six base pairs upstream of the TATA box define polarity of transcription. (a) Diagram of plasmid constructs based on pINIT with derivatives of the ef1α promoter in which the six nucleotides found upstream (pUP-INV) or downstream (pDO-INV) of the TATA box in the natural ef1α promoter are present as inverted repeats flanking the TATA box. (b) In vitro transcription assays using pUP-INV and pDO-INV. Transcription products were detected by using the T3 or T7 sequencing primers. (c) EMSAs using TFB and TBP on double-stranded oligonucleotides corresponding to the pUP-INV or pDO-INV TATA box and flanking sequences.

Figure 3

Polarity is defined at the level of ternary complex formation. (a) DNase I footprinting of TBP/TFBc complex on pEF1. Reaction mixtures contained free probe or 50 ng of TBP and 50 ng of TFBc where indicated. The TATA box is shown by a filled rectangle, and protected sequences are indicated at the right of each gel. Asterisks indicate protein-induced hypersensitivity to DNase I cleavage. (b) DNase I footprinting as in a on pFLIP2.6. (c) Summary of DNase I footprinting data. The sequences of the inserts in pEF1 and pFLIP2.6 are shown with the regions protected from DNase I cleavage boxed. The TATA box is indicated by a solid black bar. Sites of protein-induced hypersensitivities are indicated by asterisks.

Figure 4

The BRE is highly conserved and defines transcription polarity on other archaeal promoters. (a) Alignment of characterized Sulfolobus promoter sequences. R = purine; Y = pyrimidine; lack of conservation is indicated by a horizontal bar. (b) In vitro transcription assays using plasmid constructs with oligonucleotides corresponding to the wild-type S. shibatae 16S rRNA gene TATA box and flanks (16S WT) or with the 6 base pairs flanking the TATA box swapped (16S 6FL). The RNA specifically initiating at the T6 start site in a factor-dependent manner is shown by a filled arrowhead. The open arrowheads indicate factor-independent initiation by the RNAP; see Fig. 1 for details. (c) In vitro transcription assays using plasmid constructs with oligonucleotides corresponding to the wild-type S. shibatae T6 gene TATA box and flanks (T6 WT) or with the 6 base pairs flanking the TATA box swapped (T6 6FL). The RNA specifically initiating at the T6 start site in a factor-dependent manner is shown by a filled arrowhead.

Figure 5

The orientation of the archaeal TBP/TFB/DNA complex is the same as that of the eukaryal ternary complex. (a) Amino acid sequence of helix BH5′ and preceding residue in human TFIIB and archaeal TFB. The T278 residue is indicated with an asterisk. (b) EMSAs employing double-stranded oligonucleotides corresponding to the wild-type ef1α promoter (EFA-3) or ef1α promoter with an A⋅T to G⋅C substitution 3 base pairs upstream from the TATA box (EFG-3). Reaction mixtures were incubated with 20 ng of TBP and the indicated amount of TFB or TFB (T278A). (c) EMSAs employing double-stranded oligonucleotides corresponding to the wild-type T6 promoter (T6A-3) or T6 promoter with an A⋅T to G⋅C substitution 3 base pairs upstream from the TATA box (T6G-3). Reaction mixtures were incubated with 20 ng of TBP and the indicated amount of TFB or TFB (T278A). (d) Partial sequence of probes used in UV crosslinking experiments. The positions of 5-bromodeoxyuridine (BrdUrd) substitutions for thymidine are indicated by X and underlined. (e) The presence of BrdUrd substitutions upstream of the TATA box allows UV-mediated crosslinking of TFB to DNA. Five femtomoles (≈20,000 cpm) of double-stranded oligonucleotide containing radiolabeled X-UP (lanes 1–4) or X-DO (lanes 5–8) were incubated with 20 ng of the indicated proteins and irradiated as described in Materials and Methods prior to electrophoresis on an SDS/12% polyacrylamide gel. The position of TFB crosslinked to the X-UP-containing oligonucleotide probe is indicated by an arrow; the faint signal below TFB corresponds to a proteolytic fragment of TFB, as confirmed by Western blotting (data not shown).