RNA–protein interactions at/near the pre-mRNA branch site in the yeast Bact complex and the pivotal role of A30 of U2 snRNA. (A) Schematic of the region upstream and downstream from the branch site (A501), as found in the cryo-EM structure of the yeast Bact complex (pdb: 5GM6; EM density: EMD-9524), containing an ensemble of natural pre-mRNA (Yan et al. 2016). (Black dots) 5′ phosphates. (Blue dots) The homologous nucleotides of the actin pre-mRNA found protected or weakly accessible on their W/C positions (this manuscript, Supplemental Fig. S4). (Boxed nucleotides) Suboptimal base-pairing due to the large distance between the pairing partners. Amino acid side chain interactions are shown with arrows, with the protein only indicated when it is not Hsh155. The branch site is nucleotide A501 of the pre-mRNA; the U2/BS helix and the extended U2/BS helix are indicated. (B) Path of the extended U2/BS helix. The electrostatic surface of the cavity harboring the extended U2/BS helix and formed by Prp11 (U2 snRNA contacts) and Hsh155 (intron contacts). Intron sequences are not conserved and hence the backbone contacts are the major determinants for the paths of the RNAs. Charges are shown as heat maps, with blue for positive and red for negative potential (±5 kT). (C) The path of the single-stranded polypyrimidine tract across the superhelical α-solenoid structure of the Hsh155 HEAT repeats. Charges are as in B. (D) A close-up view of the counter-clockwise rotational movement of the region of U2 snRNA downstream from U2–A30 (red) encompassing the U2/BS helix and extended U2/BS helix. The coordinates from the cryo-EM structures from the Bact (5GM6, Yan et al. 2016) to C (5GMK, Wan et al. 2016a) to C* (5WSG, Yan et al. 2017) were aligned on U5 snRNA and only U2 snRNA (yellow) is shown. The U2/U6 helix Ia is behind the plane of the drawing. The pivotal U2–A30 is in red and is part of the flexible linker 1, AAGU (nts 30–33 of yeast U2 snRNA; Fig. 2B).
