Rex1 function requires both the DEDD domain and flanking regions. (A) Domain organization of the yeast Rex enzymes and Rrp6. Protein lengths (amino acid residues) are indicated. (B) Western analysis of Rex1 fusion proteins. Rex1 was expressed either from the integrated rex1-TAP allele or from plasmids encoding the wild-type protein or domain swap mutants containing the DEDD domain from either Rex2 or Rex3. (C) Ethidium-stained RNA gels showing the relative mobility of 5S rRNA species from a rex1Δ mutant expressing either wild-type Rex1 or the domain swap mutants. (D) Plasmid shuffle assay comparing wild-type and rex1 domain swap mutants. (E) Threaded model of the DEDD domain of Rex1 (residues 227–372) with bound oligoribonucleotide, derived from the Pan2/RNA structure (PDB 6R9M). Side chains of residues implicated in catalysis or substrate binding are shown. (F) Western analysis and 5S rRNA northern analysis of wild-type and rex1 point mutants. (G) Plasmid shuffle assay of the rex1 point mutants. (H) Western analysis of wild-type and rex1 deletion mutants. (I) Plasmid shuffle assay of the rex1 deletion mutants. (J) Northern analysis of 5S rRNA from the rex1 deletion mutants. Expression from low copy number, centromeric (CEN) or high copy number 2 micron (2µ) plasmids is indicated in each panel. Expression levels of mutant Rex1 proteins, normalized to Pgk1 and expressed as a percentage of the wild-type protein, are indicated beneath each lane. Values given are the mean average (av) and the mean absolute deviation (mad) of three independent replicates.
