,A Stable Upstream Stem-loop Structure Enhances Selection of the First 5′-ORF-AUG as a Main Start Co

• 作 者: Li YANG Jiang CHEN Catherine C. Y. CHANG Xin-Ying YANG Zhen-Zhen WANG Ta-Yuan CHANG Bo-Liang LI
• 机 构: State Key Lahoratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Inst
• 出 处: 生物化学与生物物理学报（英文版）
• 发表日期: 2004年4期
• 关键词: ACAT1 start codon Kozak sequence stem-loop structure

Human ACAT1 cDNA K1 was first cloned and functionally expressed in 1993. There are two adjacent in-frame AUG codons, AUG1397-1399 and AUG1415-1417, at 5′-terminus of the open reading frame (ORF,nt 1397-3049) of human ACAT1 mRNA corresponding to cDNA K1. In current work, these two adjacent inframe 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-AUG1397-1399 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-AUG1397-1399 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.