(04013) 36-4 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

Li YANG, Jiang CHEN,
Catherine C. Y. CHANG1, Xin-Ying YANG, Zhen-Zhen WANG,
Ta-Yuan CHANG1, and Bo-Liang LI*
( State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell
Biology, Shanghai Institutes for Biological Sciences,
the Chinese Academy of Sciences, Shanghai 200031, China; 1Department of Biochemistry,
Dartmouth Medical School, Hanover, NH 03756, USA)

Abstract Human ACAT1 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.

Key words ACAT1; start codon;
Kozak sequence; stem-loop structure

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Received: January 19, 2004 Accepted:
January 30, 2004
This work was supported by the grants from the Major State Basic Research Development
Program of China (No. 2002CB513000), the National High Technology Research and
Development Program of China (2001AA221061) and the National Natural Science
Foundation of China (No. 30170459, 30393112), SSTC (No. 02DJ14068), CAS to Bo-Liang
LI, and NIH grant HL36709 to Ta-Yuan CHANG
The abbreviations used are: ACAT, acyl-coenzyme A:cholesterolacyltransferase;
kb, kilobase(s); bp, base pair(s); cDNA, complementary DNA; nt, nucleotide(s);
ORF, open reading frame; CHO, Chinese hamsterovary; ER, endoplasmic reticulum;
FBS, fetal bovine serum; PBS, phosphate-buffered saline; PCR, polymerase chain
reaction; PAGE,polyacrylamide gel electrophoresis
*Corresponding author: Tel, 86-21-54921278; Fax, 86-21-54921011; Email,[email protected]