Figure 6.

Sequence alignment of the peptide encoded by exon 2 of CRS and its interaction with EF-1γ. (A) The peptide of CRS exon 2 shows sequence homology with those of human elongation factor 1β (43% over 53 residues), human EPRS (glutamyl-prolyl-tRNA synthetase) (43% over 55 residues), p18 (30% over 55 residues) and human EF-1γ (30% over 55 residues). Multiple sequence alignment of the above five proteins was prepared by CLUSTALX and the conserved residues are underlined. (B) (Left) Interactions of the peptide encoded by CRS exon 2 with the subunits of EF-1 and p18 (27) were tested by yeast two-hybrid assay. Exon 2⁺ CRS, exon 2^– CRS and exon 2 alone were expressed as hybrid proteins with LexA and the EF-1 subunits and p18 were fused to B42. A positive interaction was determined as a blue color (shown as dark color in the print) on X-gal-containing medium. (Right) The interaction of exon 2⁺ CRS with EF-1γ was tested by in vitro pull-down assay. The in vitro translated ³⁵S-labeled exon 2⁺ CRS was mixed with GST–EF-1γ or GST alone and bound to glutathione–Sepharose. The proteins eluted from the Sepharose beads were separated by SDS gel electrophoresis and the radioactive exon 2⁺ CRS was detected by autoradiography.