Alignment of sequence motifs from KREPB4, KREPB5, KREN1, KREN2, and KREN3, highlighting residues mutated in this study with outlined boxes. Amino acid numbers for each protein are indicated. Inset shows mutations examined, with arrows indicating those that showed a growth defect when exclusively expressed (see Fig. 2). Residues required for RNase III catalytic activity denoted by an open triangle. (A) The U1-like zinc finger motif (C2H2 shaded in gray) is conserved in KREPB4 but degenerates in KREPB5. (B) The RNase III signature motif depicts universally conserved catalytic Asp (gray shading) that is a Glu in both KREPB4 and KREPB5. The α-helical region responsible for dimerization with another RNase III domain to form the catalytic valley is noted. (C) C-terminal portion of RNase III motif and adjacent PUF domain. The universally conserved catalytic Glu residue is conserved in KREN1-3 but degenerates in KREPB4 and KREPB5 in this alignment. The single PUF motif (region denoted by bar) contains a conserved His residue (gray shading) in all 63 kinetoplastids examined, which is consistent with the base-stacking amino acid in known PUF domains. The Glu residue (denoted by solid triangle) in KREPB4 and KREPB5 was previously (Worthey et al. 2003) aligned with the C-terminal RNase III catalytic Glu but is consistent with being one of two base-pairing amino acids in the PUF motif in the current alignment. For alignment details, see Materials and Methods.
