R43, R44, and R52 are required for She2p association with ASH1 mRNA. (A) Amino acid sequence for She2p with amino acids N36, R43, R44, R49, R52, and R63 indicated by the black arrowheads. The black rectangle indicates the region of She2p corresponding to the predicted leucine zipper motif. (B) She2p RNA-binding activity for mutants R43K, R43A, R44K, R44A, R49K, R49A, R52K, R52A, and R63A as measured by three-hybrid assay. Strain YLM585 was transformed with the following combinations of plasmids: pRL80 (pIIIA-MS2-2-E3)/pRL445 (pGAD-c1-She2p-wt), pRL80/pRL552 (pGAD-c1-She2p-R43K), pRL80/pRL669 (pGAD-c1-She2p-R43A), pRL80/pRL553 (pGAD-c1-She2p-R44K), pRL80/pRL670 (pGAD-c1-She2p-R44A), pRL80/pRL554 (pGAD-c1-She2p-R49K), pRL80/pRL671 (pGAD-c1-She2p-R49A), pRL80/pRL555 (pGAD-c1-She2p-R52K), pRL80/pRL672 (pGAD-c1-She2p-R52A), and pRL80/pRL673 (pGAD-c1-She2p-R63A). Transformants were grown in liquid culture and processed for β-galactosidase assays.
