A stable upstream stem-loop structure enhances selection of the first 5′-ORF-AUG as a main start codon for translation initiation of human ACATl mRNA

Human ACAT 1 cDNA K1 was first cloned and functionally expressed in 1993. There are two adjacent in-frame AUG codons, AUG(1397-1399) and AUG(1415-1417), at 5'-terminus of the open reading frame (ORF, nt 1397-3049) of human ACAT 1 mRNA corresponding to cDNA K1. In current work, these two adjacent in-frame AUGs at 5'-terminus of the predicted ORF (5'-ORF-AUGs) as start codons for translation initiation of human ACAT1 mRNA were characterized in detail. Codon mutations indicated that both of these two adjacent 5'-ORF-AUGs can be selected as start codons but the first 5'-ORF-AUG(1397-1399) is a main start codon consistent with that of the predicted ORF of human ACAT1 mRNA. Further deletion and mutation analyses demonstrated that a stable upstream stem-loop structure enhanced the selection of the first 5'-ORF-AUG(1397-1399) as a main start codon, in addition to upstream nucleotide A in the -3 position, which is a key site of Kozak sequence. In addition, result of ACAT1 enzymatic activity assay showed no obvious difference between these two ACAT1 proteins respectively initiated from the two adjacent 5'-ORF-AUGs. This work showed that a stable upstream stem-loop structure could modulate the start codon selection during translation initiation of mRNAs that contain adjacent multi-5'-ORF-AUGs.