The addition of TFIIE to the growing PIC resulted in new protein density that connects TFIIF with Pol II’s stalk domain (). The resolution of this reconstruction (11 Å) was the highest obtained for any of the complexes studied, suggesting that TFIIE stabilizes the PIC. The density corresponding to TFIIE, however, was the least well-defined element according to local resolution calculations (Supplementary Fig. 4f
), which may be due to flexible connections between the WH domains predicted within the TFIIE structure6
(). One end of TFIIE associates with the stalk of Pol II by interacting with the RPB7 L45 loop (), which has been predicted to stabilize the OC and whose deletion completely abolished transcription25
. Also consistent with the positioning of TFIIE in our PIC structure, a zinc ribbon domain within the archaeal homolog of TFIIE was found to be located near the base of the stalk domain of the polymerase26
. Away from the stalk, the TFIIE density contacts the Pol II clamp domain to interact ultimately with the WH domain of TFIIF. A model of the three WH domains within TFIIE interacting with elements of the clamp head has been proposed based on crosslinking studies6
(). Although the model cannot fit the EM density perfectly, the overall path of the three tandem WH domains in the model follows the elongated TFIIE cryo-EM density and ends by directly contacting the RAP30 WH domain (). Therefore, a continuous chain of four WH domains appears to link the Pol II clamp region with the TBP-TFIIA-TFIIB-DNA subcomplex, preventing DNA from leaving the cleft.
Stabilization of the PIC in the closed conformation by TFIIE
Our 11 Å resolution reconstruction of the PIC containing TFIIE starts to reveal the major and minor grooves of the promoter DNA (), allowing us to model its path. We found that linear B-form DNA could not be accommodated into the DNA density (Supplementary Fig. 12
), requiring instead a smooth bend of 18° between positions −23 and +7 that fitted both the path and groove features of the EM density. Interestingly, a hypersensitivity region around −6 position27
locates at one of the downstream DNA-Pol II interfaces as discussed below.
We observed two protein contacts with the downstream DNA. One connection involves the 3-strand β sheet below the clamp head while the other is mediated by a 2-helix bundle at the tip of the RPB5 jaw (). Interestingly, these are the only two positively charged protein surfaces on Pol II along the path of the downstream DNA (Supplementary Fig. 12
). The INR element is sandwiched precisely between these two protein-DNA contacts, an arrangement that may be relevant in promoter melting at the correct position in the DNA. The slightly open clamp conformation seen upon DNA placement onto the cleft following TFIIF addition is likely due to the interaction of the DNA with the clamp head β sheet ( and Supplementary Fig. 11b,c
Whereas the spacing between the TATA box and the TSS can vary between species, the region within promoter DNA that is melted during transcription initiation is ~20 bp downstream of TATA28
. We inferred the approximate position of flexible elements within TFIIB and TFIIF by docking their crystal structures as rigid bodies within our cryo-EM density. Importantly, we find that both the TFIIB linker helix and the TFIIF arm domain align with the promoter melting start site (Supplementary Fig. 13
). This arrangement is consistent both with the proposed role of the linker helix of TFIIB in promoter opening12
and with the crosslinking of the arm domain of RAP74 to the TFIIB linker near the active site29,30
, as well as with the suppression of the TSS defect of TFIIB mutations by a mutant within the arm domain of TFIIF31
. In our rigid-body fitting, the linker helix of TFIIB overlaps with the DNA in our model, suggesting a rearrangement of the helix relative to the clamp domain at this stage in the PIC assembly. Finally, the tip of the TFIIF arm domain contains seven positively charged residues, whereas four positively charged residues are present on the side of the TFIIB linker helix that faces the DNA (Supplementary Fig. 13
). The juxtaposition of these domains within the melting start site is consistent with their direct role in DNA interactions.
The structural features of our Pol II based PIC model are likely conserved with Pol I and Pol III, the two other RNA polymerases in eukaryotes. A side-by-side comparison of our Pol II-based PIC model with a cryo-EM structure of native Pol III agrees with this hypothesis32
(Supplementary Fig. 14