ISSN 0582-9879 Acta Biochim et Biophysica Sinica 2004, 36(4):259-268 CN 31-1300/Q

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;
1 Department 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, 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.

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

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
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