Tight binding of Gdown1 represses RNA polymerase II (Pol II) function in a manner that is reversed by Mediator, but the structural basis of these processes is unclear. Although Gdown1 is intrinsically disordered, its Pol II interacting domains were localized and shown to occlude transcription factor IIF (TFIIF) and transcription factor IIB (TFIIB) binding by perfect positioning on their Pol II interaction sites. Robust binding of Gdown1 to Pol II is established by cooperative interactions of a strong Pol II binding region and two weaker binding modulatory regions, thus providing a mechanism both for tight Pol II binding and transcription inhibition and for its reversal. In support of a physiological function for Gdown1 in transcription repression, Gdown1 co-localizes with Pol II in transcriptionally silent nuclei of early Drosophila embryos but re-localizes to the cytoplasm during zygotic genome activation. Our study reveals a self-inactivation through Gdown1 binding as a unique mode of repression in Pol II function.
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The cryo-EM density maps of Pol II(G) have been deposited in the Electron Microscopy Data Bank under accession EMD-7997. Additional source data are available from the corresponding author upon reasonable request.
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This study was supported by National Institutes of Health Grants CA202245 and DK071900 (R.G.R.), GM067167 (F.J.A.), GM103314 and GM109824 (B.T.C.), GM067167 (F.J.A.), GM083960 and GM109824 (A.S.), and Tri-I Stem Cell Initiative Grant 2014-21 (R.G.R.). We thank Z. Xue for preparation of nuclear extracts and S. Malik for discussions and critical reading of the manuscript.
The authors declare no competing interests.
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Integrated supplementary information
a, SDS-PAGE analysis (sliver staining) of purified Pol II(G). b, Observation of individual Pol II(G) under cryo-EM conditions with a K2 direct electron detector. c, 2D classification result from the full Pol II(G) data set. d, Computational sorting of cryo-EM particle images and map reconstructions. Three-dimensional classification helps to select the homogenous particles. Only one class was selected and further subjected to movie processing and 3D Auto-Refinement. e, Local resolution of Pol II(G) cryo-EM map. The Pol II(G) ~4-Å cryo-EM map is colored by local resolution and shown as a surface (top) and slice through (bottom). f, FSC plot and resolution estimation using the gold-standard 0.143 criterion for the cryo-EM map, as well as the FSC plot for the Pol II(G) model versus the Pol II(G) map. g, Angular distribution plot for the Pol ll(G) cryo-EM image set.
Supplementary Figure 2 Gdown1 interaction sites overlap with TFIIF and TFIIB contact sites on Pol II.
a, Position of the TFIIF dimerization domain relative to Gdown1 density. K345 (shown in green) in the RPB2 lobe domain was cross-linked to K282 in the Gdown1 C-terminal region. b, Position of the TFIIB B-core domain relative to Gdown1 density. K427 (shown in green) in the RPB2 protrusion domain was cross-linked to the Gdown1 C-terminal region. c, Gdown1 C-terminal region interaction sites around the RPB1 clamp loops. Cross-linked lysines in the RPB1 clamp (K213) and rudder (K331) domains are shown in green. TFIIB is shown in yellow ribbon. d, Pol II binding assay. 35S-labeled TFIIB was incubated with Pol II or Pol II(G) and subjected to immunoprecipitation with anti-CTD antibody (8W16). Bound proteins were analyzed by SDS-PAGE and autoradiography. e, In vitro transcription assay with premelted promoter template. In vitro transcription assay was performed as described in Fig. 3d, except that the reaction time was reduced to 5 min. Note that the use of a premelted template bypasses the normal absolute TFIIF requirement for transcription, although transcription is still enhanced by TFIIF. The bands represent short transcripts resulting from specific initiation followed by Pol II pausing and/or premature termination.
a, Schematic of full-length Gdown1, N-terminal (1–180), C-terminal (181–368), and C-terminal deletion mutants and summary of Pol II binding and in vitro transcription assays. b, Binding of Gdown1 fragments to Pol II. Immobilized GST or GST-Gdown1 proteins were incubated with HeLa nuclear extract in buffer C containing 0.1 or 0.3 M KCl and 0.1% NP-40. After washing with buffer C containing 0.1 or 0.3 M KCl and 0.1% NP-40, bound proteins were analyzed by immunoblot. c–e, In vitro transcription assays. Reactions contained 10 ng of TBP, 10 ng of TFIIB, 25 ng of TFIIF, 10 ng of TFIIEα, 5 ng of TFIIEβ, 50 ng of Pol II or Pol II(G), and the indicated amounts of His-tagged full-length Gdown1 or C-terminal deletion fragments. Reactions were incubated at 30 °C for 1 h, and purified RNA products were resolved by PAGE. f, Pol II binding assay. Full-length Gdown1 and C-terminal deletion fragments were incubated with Pol II in buffer C containing 0.1 M KCl and 0.1% NP-40 for 1 h and subjected to immunoprecipitation with anti-CTD antibody (8W16). Bound proteins were analyzed by immunoblot. g, h, Pol II binding assay. Immobilized GST and GST-Gdown1 proteins were incubated with HeLa nuclear extract in buffer C containing 0.1 or 0.3 M KCl and 0.1% NP-40. After washing, bound proteins were analyzed by immunoblot. i, Pol II binding assay. Immobilized GST and GST-Gdown1 proteins were incubated with purified Pol II in buffer C containing 0.1 M KCl and 0.1% NP-40. After washing, bound proteins were analyzed by immunoblot. j, Schematic of the GST-fused Gdown1 fragments that were used in g–i. Amino acid residues (L303/4) that were changed to alanine by site-directed mutagenesis are shown in a white triangle.
a, Pol II binding assay. Full-length Gdown1 and the indicated N-terminal deletion mutant fragment were incubated with Pol II and subjected to immunoprecipitation with anti-CTD antibody (8W16). Bound proteins were analyzed by immunoblot. b, EMSA with wild-type and N-terminal deletion mutant proteins. An end-labeled adenovirus ML promoter (–40 to +20) probe was incubated with 10 ng of TFIIB, 10 ng of TBP, 50 ng of TFIIF, and 50 ng of Pol II or Pol II(G). Indicated amounts of Gdown1 proteins were added to reactions. Reactions were incubated at 30 °C for 40 min and resolved by native PAGE. c, Location of Gdown1 functional domains determined by integrative modeling. C-TIR was found to exist as an overlapping region between the region (216–314) shown in dark green and the region (300–335) shown in light green. d, N-TIR location on Pol II relative to the Med2 position in the yeast Pol II–Mediator complex. A cross-link that was detected between Med2 and the RPB3 acidic region is shown in blue line.
Integrative structure determination of the architecture of Pol II(G) proceeded through four stages: (1) data gathering, (2) representing subunits and translating data into spatial restraints, (3) configurational sampling to produce an ensemble of structures that satisfies the restraints, and (4) analyzing and validating the ensemble structures and data.
a, Convergence of the model score, for the 1693 good-scoring models; the scores do not continue to improve as more models are computed essentially independently. The error bar represents the s.d. of the best scores, estimated by repeating sampling of models 100 times. b, Distribution of model scores for model samples 1 (red) and 2 (blue), comprising the 1693 good-scoring structures. c, Three criteria for determining the sampling precision (y-axis), evaluated as a function of the RMSD clustering threshold (x-axis). First, the P value is computed using the chi-squared test for homogeneity of proportions (red dots). Second, an effect size for the chi-squared test is quantified by Cramer’s V value (blue squares). Third, the population of models in sufficiently large clusters (containing at least ten models from each sample) is shown as green triangles. The vertical dotted black line indicates the RMSD clustering threshold at which three conditions are satisfied (P > 0.05, Cramer’s V < 0.10, and population of clustered models > 0.80), thus defining the sampling precision of 18.6 Å. d, Populations of sample 1 and 2 models in the two clusters obtained by threshold-based clustering using the RMSD threshold of 23.6 Å. The cluster contains 96.8% of the models. Cluster precision is shown for the cluster. The precision of the cluster defines the model precision. e, Euclidean Cα–Cα distance statistics for each cross-link in the cluster. The cross-links are sorted by average distance (ordinate axis). The error bars represent the s.d. of the distance across all models in the clusters. (Inset, Euclidean Cα–Cα distance distributions of all measured cross-links in the ensemble of solutions for the cluster. The y axis provides the normalized number of cross-links that were mapped onto the model. The dashed red line denotes the expected maximum reach of a cross-link.).
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Jishage, M., Yu, X., Shi, Y. et al. Architecture of Pol II(G) and molecular mechanism of transcription regulation by Gdown1. Nat Struct Mol Biol 25, 859–867 (2018). https://doi.org/10.1038/s41594-018-0118-5
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