Transcription by RNA polymerase II requires the assembly of the general transcription factors at the promoter to form a pre-initiation complex. The general transcription factor TF (transcription factor) IIB plays a central role in the assembly of the pre-initiation complex, providing a bridge between promoter-bound TFIID and RNA polymerase II/TFIIF. We have characterized a series of TFIIB mutants in their ability to support transcription and recruit RNA polymerase II to the promoter. Our analyses identify several residues within the TFIIB zinc ribbon that are required for RNA polymerase II assembly. Using the structural models of TFIIB, we describe the interface between the TFIIB zinc ribbon region and RNA polymerase II.

Introduction

The recruitment of pol II (RNA polymerase II) to the promoter of a gene requires the prior recognition of core promoter sequence elements by the GTFs (general transcription factors) (reviewed in [13]). TF (transcription factor) IID recognizes the TATA box, when present within the promoter, via its TBP (TATA-box-binding protein) subunit. TFIIB can then assemble and stabilizes the interaction of TFIID with the promoter (reviewed in [4]). TFIIB can also make sequence-specific contact with the core promoter DNA [58]. The assembly of TFIIB at the promoter is absolutely required for the recruitment of pol II, which enters the forming PIC (pre-initiation complex) along with TFIIF. TFIIE and TFIIH then enter and complete the complex. Several studies have suggested the existence of a preformed pol II holoenzyme complex that contains several of the GTFs in addition to other factors (reviewed in [9,10]).

The GTF TFIIB plays a critical role in the PIC [4]. TFIIB contains a zinc ribbon at the N-terminus and a C-terminal core domain composed of α-helices that form two direct repeats [1113]. The crystal structure of the TFIIB core domain–TBP–promoter complex suggests that, if the N-terminus of TFIIB were included, it would project downstream towards the region of transcription initiation [14,15]. In between the zinc ribbon and core domain lies the most conserved region of TFIIB, termed the B-finger and a linker region [1618]. Although intact TFIIB has evaded detailed structural analysis, a low-resolution structure of intact TFIIB has been observed as a complex with pol II [16]. In this structure, the B-finger lies within the pol II catalytic centre and, when assembled at the promoter, is close to the region of transcription initiation. This is consistent with previous observations that mutations within the B-finger of TFIIB can cause alterations in the start site of transcription [1924].

The zinc ribbon of TFIIB is required for the recruitment of pol II to the promoter [22,2530]. In our studies, we have generated a selection of individual amino acid substitutions within the zinc ribbon region of TFIIB. Using transcription assays, coupled with analysis of promoter complex assembly, we have characterized a surface within the TFIIB zinc ribbon that is required for the recruitment of pol II to the promoter.

Residues within the zinc ribbon of TFIIB that are critical in transcription

We initiated a mutagenesis study of a selection of highly conserved residues at the TFIIB N-terminus in order to probe the function of this region of TFIIB in transcription. Figure 1(A) shows a schematic representation of TFIIB, with the amino acid sequence of the zinc ribbon and B-finger shown below. The residues indicated by an arrow were mutated to alanine within full-length TFIIB. The recombinant proteins were expressed in Escherichia coli, purified and analysed by SDS/PAGE with Coomassie Blue staining (Figure 1B). The TFIIB mutants were then added to a HeLa cell nuclear extract that had been depleted of endogenous TFIIB by immunoaffinity chromatography. The supplemented extract was used in an in vitro transcription assay with the AdML (adenovirus major late) promoter in the presence of the activator GAL4–VP16 (viral protein 16) (Figure 2A). Compared with wild-type TFIIB, the TFIIB mutants C34A, G30A, D31A, V40A and W52A showed a severe defect in supporting transcription. TFIIB E25A and E36A showed a moderate impairment, whereas F55A and V67A supported higher levels of transcription.

TFIIB zinc ribbon mutants

Figure 1
TFIIB zinc ribbon mutants

(A) Schematic representation of human TFIIB (hTFIIB) showing the zinc ribbon (Zn), B-finger (BF), linker and direct repeats (arrows). The N-terminal sequence from H. sapiens is shown below, and the residues subject to mutation are shown by arrows. (B) Coomassie Blue-stained SDS/PAGE gels showing recombinant wild-type TFIIB (wtIIB) and the mutants (500 ng/lane). Molecular-mass markers (kDa) are shown on the left.

Figure 1
TFIIB zinc ribbon mutants

(A) Schematic representation of human TFIIB (hTFIIB) showing the zinc ribbon (Zn), B-finger (BF), linker and direct repeats (arrows). The N-terminal sequence from H. sapiens is shown below, and the residues subject to mutation are shown by arrows. (B) Coomassie Blue-stained SDS/PAGE gels showing recombinant wild-type TFIIB (wtIIB) and the mutants (500 ng/lane). Molecular-mass markers (kDa) are shown on the left.

Definition of TFIIB zinc ribbon residues required for transcription and recruitment of pol II

Figure 2
Definition of TFIIB zinc ribbon residues required for transcription and recruitment of pol II

(A) In vitro transcription analysis of the TFIIB zinc ribbon mutants. A sample of 50 or 100 ng of wild-type TFIIB (wtIIB) or each mutant indicated was added to a HeLa cell nuclear extract that had been depleted of TFIIB along with 50 ng of GAL4-(1–94)–VP16 and the reporter G9MLCAT [7,33]. Transcripts were analysed by primer extension with a radiolabelled CAT (chloramphenicol acetyltransferase) primer, subject to denaturing electrophoresis and visualized by autoradiography. The in vitro transcription method was as described previously [21,34]. (B) EMSA to analyse the formation of a pol II/TFIIF–TFIIB–TBP–AdML promoter complex. Where indicated the reactions contained 4 ng of TBP, 50 ng of pol II, 10 ng of TFIIF and 25 or 50 ng of TFIIB. Complexes were resolved by non-denaturing electrophoresis and detected by autoradiography. EMSAs were performed as described previously [31,35]. FP, free probe; TB, TFIIB–TBP–promoter complex; TBpolF, pol II–TFIIF–TFIIB–TBP–promoter complex; wtTFIIB, wild-type TFIIB.

Figure 2
Definition of TFIIB zinc ribbon residues required for transcription and recruitment of pol II

(A) In vitro transcription analysis of the TFIIB zinc ribbon mutants. A sample of 50 or 100 ng of wild-type TFIIB (wtIIB) or each mutant indicated was added to a HeLa cell nuclear extract that had been depleted of TFIIB along with 50 ng of GAL4-(1–94)–VP16 and the reporter G9MLCAT [7,33]. Transcripts were analysed by primer extension with a radiolabelled CAT (chloramphenicol acetyltransferase) primer, subject to denaturing electrophoresis and visualized by autoradiography. The in vitro transcription method was as described previously [21,34]. (B) EMSA to analyse the formation of a pol II/TFIIF–TFIIB–TBP–AdML promoter complex. Where indicated the reactions contained 4 ng of TBP, 50 ng of pol II, 10 ng of TFIIF and 25 or 50 ng of TFIIB. Complexes were resolved by non-denaturing electrophoresis and detected by autoradiography. EMSAs were performed as described previously [31,35]. FP, free probe; TB, TFIIB–TBP–promoter complex; TBpolF, pol II–TFIIF–TFIIB–TBP–promoter complex; wtTFIIB, wild-type TFIIB.

TFIIB zinc ribbon mutants that fail to recruit pol II

These same TFIIB mutants were then assessed for their ability to assemble with TBP and recruit pol II/TFIIF to the promoter using an EMSA (electrophoretic mobility-shift assay) (Figure 2B). All of the TFIIB mutants were able to assemble into a TFIIB–TBP–AdML promoter complex. However, the TFIIB mutants C34A, G30A, D31A and V40A were defective in pol II/TFIIF recruitment, whereas the other mutants supported pol II/TFIIF recruitment to a level similar to that observed with wild-type TFIIB. Thus the defects in transcription observed with the C34A, G30A, D31A and V40A TFIIB mutants can be explained, at least in part, by their failure to recruit pol II to the promoter. Co-immunoprecipitation and affinity chromatography analyses revealed that the defective TFIIB mutants maintained their ability to interact with both subunits of TFIIF, but were unable to interact directly with pol II (results not shown).

A surface at the N-terminus of TFIIB that contacts pol II

Tubon et al. [29] analysed charged amino acids within the TFIIB zinc ribbon that are required for transcription and pol II assembly, finding a role for residues Asp26, Asp31, Asp43 and Arg44. Where comparable, our results are in agreement with their findings, specifically that TFIIB residue Asp31, but not Glu25, is required for assembly of pol II at the promoter. Superimposing the residues defined here along with those described by Tubon et al. [29] on to the previously solved NMR structure of the human TFIIB zinc ribbon reveals a discrete surface that is required to contact pol II (Figure 3A; four images sequentially rotated in 90° increments). The three residues (other than the zinc-co-ordinating cysteine) that are defective in poll II recruitment and pol II binding (Gly30, Asp31 and Val40; shown in orange) are clustered together on the surface of the triple β-strand. Residues defined in the recent study of Tubon et al. [29] as critical in the pol II interaction are highlighted in yellow. Taken together, these residues form a band that crosses one surface of the TFIIB zinc ribbon. Residues mutated in this work and previous studies that do not affect the interaction with or recruitment of pol II are shown in blue (dark and light respectively [4,21,28,31]). These results define an interface within the zinc ribbon of TFIIB that lies on one surface and encompasses each of the three β-strands of the zinc ribbon motif.

TFIIB–pol II contacts

Previous crystallographic and biochemical studies have analysed the association of yeast pol II with TFIIB, finding that the zinc ribbon domain contacts the dock domain of pol II subunit Rpb1 between residues 409 and 419 (reviewed in [4]). Figure 3(B) shows the structures of the pol II Rpb1 subunit (green) and N-terminus of TFIIB (blue) from Bushnell et al. [16]. The region of Rpb1 that contacts TFIIB is highlighted in yellow and, where possible, we have highlighted (in red) residues that are required for contact with Rpb1. Gly30 is absent from this structure, but residue 29 is labelled to help predict a likely location. Significantly, in large part, these residues lie close to the site of Rpb1 contact with TFIIB. This model was originally based on the NMR structure of human TFIIB, but contains the two-amino-acid insertion that is specific to yeast TFIIB (between human residues 20 and 21). In addition, although human Gly30, Asp31, Val40 and Arg44 (as lysine) are conserved in yeast, human TFIIB residue Asp26 is not. It is possible that these differences may account for the imperfect alignment of the two interaction surfaces.

Modelling the TFIIB–pol II interface

Figure 3
Modelling the TFIIB–pol II interface

(A) Four sequential views (at approx. 90° rotation per view) of the human TFIIB N-terminus are shown. Residues mutated in our studies that are defective in interaction with pol II are shown in orange. Residues from the study of Tubon et al. [29] are shown in yellow. TFIIB residues studied here and previously [21,28,31,33], that do not affect the interaction with pol II are shown in blue (dark and light respectively). (B) The structure of pol II (Rpb1; green) complexed with the TFIIB N-terminus (blue) from Bushnell et al. [16] is shown. The TFIIB-interaction surface in Rpb1 is shown in yellow. The site of residues in TFIIB that are critical in association with pol II are shown in red. The zinc atom in TFIIB is shown in pale blue. The image was generated using PyMOL (DeLano Scientific; http://pymol.sourceforge.net/). Single-letter amino acid codes are used.

Figure 3
Modelling the TFIIB–pol II interface

(A) Four sequential views (at approx. 90° rotation per view) of the human TFIIB N-terminus are shown. Residues mutated in our studies that are defective in interaction with pol II are shown in orange. Residues from the study of Tubon et al. [29] are shown in yellow. TFIIB residues studied here and previously [21,28,31,33], that do not affect the interaction with pol II are shown in blue (dark and light respectively). (B) The structure of pol II (Rpb1; green) complexed with the TFIIB N-terminus (blue) from Bushnell et al. [16] is shown. The TFIIB-interaction surface in Rpb1 is shown in yellow. The site of residues in TFIIB that are critical in association with pol II are shown in red. The zinc atom in TFIIB is shown in pale blue. The image was generated using PyMOL (DeLano Scientific; http://pymol.sourceforge.net/). Single-letter amino acid codes are used.

In contrast with mutations within the zinc ribbon, several mutations within the B-finger do not quantitatively affect the interaction between TFIIB and pol II (reviewed in [4]; see Figure 3A). Mutations within the TFIIB B-finger frequently cause either a shift in the transcription start site or a block in transcription that occurs after recruitment of pol II to the promoter [4]. The TFIIB B-finger projects into the catalytic centre of pol II ([1618]; see Figure 3B), suggesting that the effects of B-finger substitutions might have direct effects on catalytic activity. Consistent with this notion, B-finger substitutions have been found to affect promoter clearance [32]. Thus TFIIB elicits two temporally spaced effects on pol II; first by recruitment of pol II to the promoter and second by modulation of pol II activity.

Transcription: A Biochemical Society Focused Meeting held at the University of Manchester, U.K., 26–28 March 2008 as part of the Gene Expression and Analysis Linked Focused Meetings. Organized and Edited by Stefan Roberts (Manchester, U.K.) and Robert White (Beatson Institute, Glasgow, U.K.).

Abbreviations

     
  • AdML

    adenovirus major late

  •  
  • EMSA

    electrophoretic mobility-shift assay

  •  
  • GTF

    general transcription factor

  •  
  • PIC

    pre-initiation complex

  •  
  • pol II

    RNA polymerase II

  •  
  • TBP

    TATA-box-binding protein

  •  
  • TF

    transcription factor

  •  
  • VP16

    viral protein 16

We thank the Wellcome Trust for funding our work. S.G.E.R. is a Wellcome Trust Senior Research Fellow.

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