Figure 6  Structure-guided alanine scan of Rpo26. (A) Annotated structure of Rpo26 (from pdb 1I3Q) highlighting atomic interactions (dashed lines) of selected side chains and main chain atoms (depicted as stick models with beige carbons). Eight residues were targeted for alanine scanning: Arg79, Glu89, Arg97, Glu124, Arg135, Arg136, Asp145, and Glu150. The lethal RPO26-Ala alleles are indicated on the right. (B) rpo26∆ complementation. Yeast strain rpo26Δ p(URA3 CEN RPO26) was transformed with 2-μ LEU2 TPI1-RPO26 plasmids bearing wild-type RPO26, RPO26-(78-155), or the indicated RPO26-Ala mutants. Leu+ transformants were selected at 30° on agar medium containing 5-FOA (1.0 mg/ml) and aliquots of serial 10-fold dilutions of the strains with the specified genotypes were spotted on YPD agar medium. The plates were photographed after incubation for 2 d at 37°, 3 d at 30°, 5 d at 25°, or 7 d at 18°. (C) tgs1∆ suppression. tgs1Δ cells were transformed with a CEN LEU2 plasmid bearing wild-type TGS1 (positive control), an empty 2-μ LEU2 TPI1 vector (negative control), or 2-μ LEU2 TPI1-RPO26 plasmids bearing wild-type RPO26 or the indicated RPO26-Ala mutants. Leu+ transformants were selected at 30° and then tested for growth at 18° by spotting serial 10-fold dilutions of liquid cultures (grown at 30° in SD-Leu medium) on SD-Leu agar plates. The plates were photographed after incubation for 7 d at 18°.