A stable upstream stem-loop structure enhances selection of the first 5'-ORF-AUG as a main start codon for translation initiation of human ACAT1 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 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-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.
Yang
Li
L
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences.
Chen
Jiang
J
Chang
Catherine C Y
CC
Yang
Xin-Ying
XY
Wang
Zhen-Zhen
ZZ
Chang
Ta-Yuan
TY
Li
Bo-Liang
BL
D020121:5' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D018387:Codon, Initiator; D006224:Cricetinae; D003412:Cricetulus; D004252:DNA Mutational Analysis; D017353:Gene Deletion; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D002785:Sterol O-Acyltransferase / Q000235:genetics*
D020121:5' Untranslated Regions; D018387:Codon, Initiator; D012333:RNA, Messenger; D002785:Sterol O-Acyltransferase; C493492:sterol O-acyltransferase 1