Figure 2.

Point mutations in the conserved CCCH motif are inconsistent with a disulfide bond, but are consistent with metal binding. As previously reported, simultaneously mutating the three conserved Cys residues to Ser causes a slow growth phenotype (A) and stabilization of his3-nonstop mRNA (B). The single C47S mutation has a similar, but slightly less severe defect, which can be seen after 6 days on 5-FOA plates in the middle panel. Shorter incubations (e.g. 2 days; left panel) show that the rrp44–C52S allele also has a significant but less severe defect. For panel A a rrp44Δ strain with a low copy plasmid with the wild-type RRP44 and URA3 genes was transformed with a second plasmid with a LEU2 gene and the indicated RRP44 gene (none, wild-type, rrp44–CR3, rrp44–C47S, rrp44–C52S, rrp44–C55S, or rrp44–H184A). Transformants were selected and then serially diluted and spotted on 5FOA containing plates that select for cells that have lost the URA3 plasmid or SC–URA–LEU plates. For Panel B, the colonies that grew on 5FOA were then transformed with a reporter plasmid that contains the his3-nonstop and URA3 genes. Transformants were selected and then serially diluted and spotted on SC–URA–LEU–HIS plates or SC–URA–LEU plates.