Non-AUG initiation of AGAMOUS mRNA translation in Arabidopsis thaliana

The MADS box organ identity gene AGAMOUS (AG) controls several steps during Arabidopsis thaliana flower development. AG cDNA contains an open reading frame that lacks an ATG triplet to function as the translation initiation c odon, and the actual amino terminus of the AG protein remains uncharacteriz ed. We have considered the possibility that AG translation can be initiated at a non-AUG codon. Two possible non-AUG initiation codons, CUG and ACG, a re present in the 5' region of AG mRNA preceding the highly conserved MADS box sequence. We prepared a series of AG genomic constructs in which these codons are mutated and assayed their activity in phenotypic rescue experime nts by introducing them as transgenes into ag mutant plants. Alteration of the CTG codon to render it unsuitable for acting as a translation initiatio n site does not affect complementation of the ag-3 mutation in transgenic p lants. However, a similar mutation of the downstream ACG codon prevents the rescue of the ag-3 mutant phenotype. Conversely, if an ATG is introduced i mmediately 5' to the disrupted ACG codon, the resulting construct fully com plements the ag-3 mutation. The AG protein synthesized in vitro by initiati ng translation at the ACG position is active in DNA binding and is of the s ame size as the AG protein detected from floral tissues, whereas AG polypep tides with additional amino-terminal residues do not appear to bind DNA. Th ese results indicate that translation of AG is initiated exclusively at an ACG codon and prove that non-AUG triplets may be efficiently used as the so le translation initiation site in some plant cellular mRNAs.