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. 1997 Jul 8;94(14):7150-5.
doi: 10.1073/pnas.94.14.7150.

RAP74 induces promoter contacts by RNA polymerase II upstream and downstream of a DNA bend centered on the TATA box

D Forget  1 , F Robert, G Grondin, Z F Burton, J Greenblatt, B Coulombe
Affiliations

RAP74 induces promoter contacts by RNA polymerase II upstream and downstream of a DNA bend centered on the TATA box

D Forget et al. Proc Natl Acad Sci U S A. .

Abstract

RAP74, the large subunit of transcription factor IIF, associates with a preinitiation complex containing RNA polymerase II (pol II) and other general initiation factors. We have mapped the location of RAP74 in close proximity to promoter DNA at similar distances both upstream and downstream of a DNA bend centered on the TATA box. Binding of RAP74 induces a conformational change that affects the position of pol II relative to that of the DNA. This reorganization of the preinitiation complex minimally requires the N-terminal region of RAP74 containing both its RAP30-binding domain and another region necessary for accurate transcription in vitro. We propose a role for RAP74 in controlling the topological organization of the pol II preinitiation complex.

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Figures

Figure 1
Figure 1
Crosslinking of RAP74 and pol II along the adenovirus major late promoter. (A) Independent photoprobes that place the modified nucleotide deoxyuridine monophosphate (U) and radiolabeled nucleotides at various positions along the adenovirus major late promoter were used in crosslinking experiments. Binding reactions contained TFIIB, TFIIF (RAP74 and RAP30), pol II, and TFIIE (56- and 34-kDa subunits) in either the presence (+) or the absence (−) of TBP. The positions of RAP74 as well as those of the 145- and 180-kDa subunits of pol II are indicated. (B) Photocrosslinking experiments in the presence of TBP, TFIIB, TFIIF, pol II, and TFIIE were performed using photoprobe −39/−40 that contained either a wild-type (TATAAAA) or a mutated (TAGAGAA) TATA element. (C) Photocrosslinking experiments were performed using TBP, TFIIB, TFIIF, pol II, and TFIIE (All factors), or all of the above factors except TBP (No TBP), RAP74 (No RAP74), or pol II (No Pol II). In some cases, crosslinked polypeptides were immunoprecipitated using either antibodies directed against RAP74 (Anti-RAP74) or control antibodies (Control Ab). (D) Protein factors used in the crosslinking experiments.
Figure 2
Figure 2
Effect of RAP74 deletion mutants on the crosslinking of pol II upstream of the TATA element. (A) Functional domains of RAP74 and the sequences included in the various C-terminal deletion mutants are indicated. (B and C) Photocrosslinking experiments with either photoprobe −39/−40 (B) or −19 (C) were performed using TFIIB, RAP30, pol II, and TFIIE in the presence (+) or in the absence (−) of TBP and/or RAP74 (either full-length or various C-terminal deletion mutants). Some binding reactions were conducted in the absence of RAP74 (No RAP74). The positions of the 145- and 180-kDa subunits of pol II are indicated. (D) Summary of the photocrosslinking data shown in B and C.
Figure 3
Figure 3
(A) A typical EM field showing a preinitiation complex (arrow) assembled on the adenovirus major late promoter in the presence of TBP, TFIIB, TFIIF, pol II, and TFIIE. Two uncomplexed DNA fragments (double-headed arrow) and one free protein complex (arrowhead) are also shown. (B) Histograms of DNA lengths of promoter fragments either carrying a preinitiation complex or in the uncomplexed state.
Figure 4
Figure 4
(A) Schematic representation of the conformational change induced by RAP74 in a TBP–TFIIB–RAP30–pol II–TFIIE–promoter complex. The entry of RAP74 in the complex modifies the position of the 145-kDa pol II subunit relative to positions −39/−40 upstream of the TATA box and that of the 180-kDa pol II subunit relative to position −19 downstream of the TATA box. Because we do not yet know whether the conformational change affects the absolute position of the DNA, the polymerase, or both, double-headed arrows are used to indicate the relative movement of pol II subunits. (B) Low resolution model of a TBP–TFIIB–TFIIF–pol II–TFIIE–promoter complex. The model accounts for the direct protein–protein interactions that have been reported between the various members of the general transcription machinery (–6, 26). RAP74 is located in close proximity to the DNA on each side of a bend centered on the TATA box. RAP74, RAP30, and the 145-kDa subunit of pol II occupy a space on the same face of the DNA helix between positions −19 and −5. The placement of RAP74 in close proximity to pol145 is also consistent with the identification of a putative TFIIF interaction site in the IIc subunit (pol 140) of pol II in Drosophila (45). TFIIE34 and the 180-kDa subunit of pol II are placed on the other face of the helix between −19 and +1. The model is consistent with our EM data although no subunit structure can be resolved in the EM photo.
Figure 4
Figure 4
(A) Schematic representation of the conformational change induced by RAP74 in a TBP–TFIIB–RAP30–pol II–TFIIE–promoter complex. The entry of RAP74 in the complex modifies the position of the 145-kDa pol II subunit relative to positions −39/−40 upstream of the TATA box and that of the 180-kDa pol II subunit relative to position −19 downstream of the TATA box. Because we do not yet know whether the conformational change affects the absolute position of the DNA, the polymerase, or both, double-headed arrows are used to indicate the relative movement of pol II subunits. (B) Low resolution model of a TBP–TFIIB–TFIIF–pol II–TFIIE–promoter complex. The model accounts for the direct protein–protein interactions that have been reported between the various members of the general transcription machinery (–6, 26). RAP74 is located in close proximity to the DNA on each side of a bend centered on the TATA box. RAP74, RAP30, and the 145-kDa subunit of pol II occupy a space on the same face of the DNA helix between positions −19 and −5. The placement of RAP74 in close proximity to pol145 is also consistent with the identification of a putative TFIIF interaction site in the IIc subunit (pol 140) of pol II in Drosophila (45). TFIIE34 and the 180-kDa subunit of pol II are placed on the other face of the helix between −19 and +1. The model is consistent with our EM data although no subunit structure can be resolved in the EM photo.
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