Isolation and characterization of the 5' regulatory region of acetyl-coenzyme A carboxylase gene of the rat
Abstract
Acetyl-Coenzyme A Carboxylase (ACC; acetyl-CoA:carbon dioxide ligase (ADP forming), EC 6.4.1.2) catalyzes the first rate-limiting step in the biosynthesis of long chain fatty acids. Long term regulation of ACC in response to various physiological conditions is achieved by modulating the amount of ACC protein. Previous studies have shown that the changes in the amount of ACC protein is a direct result of alteration in the level of ACC mRNA. The present study focuses on the molecular mechanism of regulation of ACC gene expression at the transcriptional level, namely, how the temporal and tissue specific expression of ACC gene is reflected at least in part by its complex structural features at the 5$\sp\prime$ flanking region of this gene. Rat genomic DNA encoding the 5$\sp\prime$ untranslated region of ACC mRNA was isolated and the intron-exon structure was characterized. The 5$\sp\prime$ untranslated region of ACC mRNA is encoded by five exons which contain a total of 645 nucleotides. These exons scatter over 50 kilobase (Kb) of genomic DNA. The four upstream exons contribute to the untranslated region of the ACC mRNA, while the 5th exon contains the translational initiation site. Expression of exon 1 and exon 2 is under the control of two different promoters, P1 and P2 respectively, that give rise to two distinct ACC transcriptional units (pAU type and FL type). The combined actions of selective promoter utilization and alternative splicing of the five exons account for the generation of at least five variant species of ACC mRNA that we have previously described. The proximal promoter (P2) which drives transcription of the FL type mRNA lacks the typical TATA box and CAAT box and has a high percentage of GC contact. Six consensus sequences for SP1 binding are present within 150 bp upstream of the transcription initiation site of FL type mRNA. A strong positive cis-acting element composed of three symmetrical core sequences was identified and it stimulates an almost 13 fold increase in transcription. The distal promoter (P1) which produces pAU type mRNA possesses a typical TATA box and CAAT box. There exists a negative cis-acting element located at about 220 bp upstream of the transcription initiation site of the pAU type mRNA and the promoter activity of P1 is suppressed by about 70% by this cis-acting element. In addition, there exists a protein factor which specifically binds to the negative cis-element, and the binding of this factor is strongly correlated with the appearance and elevation of the pAU type mRNA.
Degree
Ph.D.
Advisors
Kim, Purdue University.
Subject Area
Biochemistry|Molecular biology
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