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. 2009 Dec 14;285(7):4595–4602. doi: 10.1074/jbc.M109.070532

FIGURE 4.

FIGURE 4.

Analysis of translation initiation function of the CUG codon. A, deletion and point mutation of CUG codon and its flanking areas were used to examine the ability of CUG to initiate translation. Lane 1, GFP control; lane 2, GFP fused with 5′-UTR and sequences coding for the N-terminal region of TGR-L; lane 3, GFP fusion construct carrying deletion at nucleotide positions 203–256; lane 4, GFP fusion construct with deletion at nucleotide positions 1–92; lane 5, GFP fusion construct in which CUG was mutated to CUC in nucleotide position 148; lane 6, GFP fusion construct in which CUG codon was mutated to AUG. extTGR, extended longer form of TGR. B, deletion of conserved sequences upstream of CUG does not affect translation initiation. Lane 1, GFP control; lane 2, GFP fusion construct that contains the 5′-UTR and sequences coding for the N-terminal region of TGR-L; lane 3, same construct, but with deletion in nucleotide positions 93–119. C, Kozak consensus sequences flanking the CUG codon are important for efficient initiation of translation. The upper panel summarizes mutations that were examined. The middle panel shows an immunoblot analysis with anti-GFP antibodies. Lysates of HEK 293 cells were used. Cells were transfected with GFP fusion constructs with mutations in the Kozak consensus sequence of the CUG codon. Lane 1, GFP control; lane 2, GFP fusion construct coding for the N-terminal part of TGR-L; lane 3, the same construct but CC was replaced with GT; lane 4, CA was replaced with TT; lane 5, GAG was replaced with CAT; and lane 6, GCC was replaced with CAT. The lower panel shows the protein loading control. D, Kozak consensus sequence of the longer form of TGR is sufficient to initiate translation at CUG codon in HEK 293 cells. Immunoblot analysis with antibodies specific for GFP is shown. Lane 1, expression of control GFP construct in HEK 293 cells; lane 2, expression of the GFP construct with AUG start codon replaced by CUG and its Kozak sequence (−8 to +6 positions relative to CUG) derived from the longer form of TGR.