Figure 4.
In vitro and in vivo mutational study of Nrd1. (A) GUAA binding by the Nrd1307–491 mutants assayed using FA. (B) GCGGGGC binding by the Nrd1307–491 mutants assayed using FA. (C) In vivo phenotypic analyses of the Nrd1 mutants. Wt Nrd1 contains non-mutated NRD1 gene, pRS415 is a negative control with empty plasmid without NRD1 gene and the other plasmids contain NRD1 point mutations as denoted. The indicated mutants were expressed episomally from pRS415 plasmids in the yeast strain with the endogenous NRD1 driven by GAL1 promoter. Mutant strains were spotted on plates containing 2% glucose and on a control galactose plate and incubated for 3 days at temperatures indicated. Growth on glucose containing plates leads to the repression of GAL1-driven wild-type Nrd1, and thus shows the functionality of the different Nrd1 mutants. The inviability of Nrd1 variants with asterisks (R384D and S423R) likely results from the insolubility of these mutants; they could not be assayed for RNA binding (see above). (D) Alignment of Nrd1307–491 from different yeast species along with the secondary structure elements and RNP motifs. Identical residues are highlighted in black, similar ones in gray. The RNP2 and RNP1 consensus sequences are shown in black boxes. Mutated residues with notable phenotype are labeled above the alignment; cross stands for lethality and no RNA binding, filled circle for thermosensitivity and significantly reduced RNA binding, and circle for variants with no defect in the phenotypic analysis but with significantly reduced RNA binding affinity.