FIGURE 2.

The cytosine-recognition code can be transferred to other PUM repeats. A, mutation of PUM repeat 2 to convert its binding specificity to recognize C7 RNA. Indicated mutations were introduced in repeat 2 (left). Protein-RNA binding measured with the yeast three-hybrid system using β-galactosidase activity is shown (right). Wild-type PUF and its cognate target RNA were included in all experiments as controls, and its relative activity was set to 1. B, mutation of PUM repeat 5 to convert its binding specificity to recognize C4 RNA. Indicated mutations were introduced in repeat 5. C, mutation of PUM repeat 6 to convert its binding specificity to recognize C3 RNA. Indicated mutations, including two different stacking residues, were introduced. D, mutation of PUM repeat 7 to convert its binding specificity to recognize C2 RNA. Indicated mutations, including two different stacking residues, were introduced. For panels A–D, the -fold increase in binding of the mutant protein to the cognate base versus the non-cognate base in the wild-type RNA is indicated above the bars. E, mutation of PUM repeat 3 to convert its binding specificity to recognize C6 RNA. Indicated mutations, including mutation of the base stacking residue of repeat 4, were introduced. For all panels, the experimental conditions and data analyses are similar to that in panel A. Error bars indicate S.D.