Extended Data Fig. 3: Related to Fig. 2. Structure of the pre-cleavage cap-snatching complex.

a-b, Comparison of pre-cleavage structure with the canonical Pol II-DSIF EC (PDB:5OIK)³³. a, The canonical position of the KOWx-4 domain is shown in pink, and the position observed in the pre-cleavage structure is shown in green. The KOWx-4 domain of DSIF SPT5 is displaced by ~22 Å and rotated by ~180° relative to the canonical conformation. b, The canonical position of the Pol II stalk is colored in dusty pink, and the stalk model from the pre-cleavage structure is depicted in gray. The Pol II stalk is moved by 20° around its base relative to the canonical conformation. c, Phosphorylated CTD of RPB1 bound to FluPol shown in sticks. The obtained cryo-EM density of the pre-cleavage structure is displayed in transparent gray. d, Phosphorylated CTD of RPB1 bound to FluPol shown in sticks. The obtained cryo-EM density of the post-cleavage structure is displayed in transparent gray. e,f, Comparison of CTD binding the prior structures^2,42 shows a similar conformation of the CTD in the CTD-binding site of FluPol. g, Cryo-EM density for the RNA within FluPol between the PB2 cap-binding and PA endonuclease domains. The density is colored by thresholds. Red corresponds 0.015, purple to 0.0075 and blue to 0.004. The endonuclease active site Mg²⁺ and Pol II polymerase active are shown in dark blue spheres. RNA within Pol II is shown and the corresponding density is shown in transparent gray. The density was restricted to areas close to the RNA in ChimeraX. The RNA is colored by heteroatom. h, Zoom in to 5′ mRNA cap(1) inside the PB2 cap-binding domain and the supporting Phenylalanine from the KOWx-4-KOW5 linker. The supporting Isoleucine260 of PB2 interacting with the 2′-Methoxygroup is highlighted as well. The density for the RNA and the linker is transparent and colored in the color of the underlying models. i, Schematic representation of the RNA and its interacting residues within the complex. The RNA is color coded by resolution using the same thresholds as in g. j, Active center of the endonuclease domain is shown. Magnesium chelating residues are show in yellow. In orange is the mutated PA E119D residue used in the structure. k,l Effect of mutation of DSIF on the stimulatory effect of DSIF on the endonuclease activity of FluPol in vitro. Representative example for the denaturing urea PAGE of an endonuclease assay is shown in k. The substrate and the product bands are labeled. Fraction of cleaved RNA (intensity of cleaved product divided by intensity of all bands, see Extended Data Fig. 1c) in dependence of factors added. Each point reflects one experimental replicate (n = 6), shown as mean ± s.d. Significance p-values were calculated using a linear mixed-effects model (substrate as a fixed effect, experimental replicate as a random effect, two-sided, no multiple testing correction). Comparison of DSIF WT and DSIF SPT5 F656A, F661A; DSIF SPT5 Δ552-563; DSIF SPT5 W535A have p-values of 0.0401; 0.0013 and <0.0001.