Figure 1. A highly conserved region in the Drosophila CTD is essential for viability and is targeted for Ser5 phosphorylation by Dm P-TEFb in vitro.

(a) Schematic of the rescue assay: Ubiquitous expression of shRNA targeting Rpb1 (UAS-Rpb1i) by the Actin-GAL4 transgene is lethal and results in no straight-winged progeny. Rescue as a result of co-expression of an Rpb1 derivative (UAS-Rpb1^WT/mut) is indicated by the presence of straight-winged adults among the progeny. (b) Four internal deletion mutants of Rpb1 were expressed under Actin-GAL4 control to test for rescue of lethality caused by ubiquitous depletion of endogenous Rpb1 (Actin-GAL4/+; UAS-Rpb1i/+). Only CTDΔ2 failed to rescue. (c) Western blot analysis of the expression of Rpb1 derivatives: Tissues were collected from late pupae derived from the same genetic cross as described in a. Late pupae were analysed because these were produced by each of the matings, including the one with CTDΔ2. Ectopic Rpb1 expression was detected with FLAG antibody. Detection of Spt5 served as a loading control. (d) Percentages of straight-winged adults from each cross. For numbers of flies examined, see Supplementary Fig. 1c. (e) Evolutionary conservation of the CTD across different species. Identical amino acids are denoted by black bars. The region encompassed by the CTDΔ2 deletion (CTD2) and recombinant protein CTD2′ (black lines, bottom) contain a highly conserved region.