Poster: Posttranscriptional Regulation
Abs #
1084: The involving of phosphorylation in distinct mechanism of translational regulation by upstream open reading frame of SAMDC gene
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Presenter: |
Park, Ky Young , plpm@sunchon.ac.kr | Authors | Park, Ky Young (A) Choi, Yu Jin (A) Pai, Hyun Sook (B) | | Affiliations: |
(A): Department of Biology, Sunchon National University, Sunchon, Chonnam 540-742, South Korea
(B): Laboratory of Plant Genomics, Korea Research Institute of Bioscience and Biotechnology, Oun-dong 52, Yusong-gu, Taejon 305-333, South Korea
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S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.4.50) is a key enzyme in the biosynthesis of the polyamines spermidine and spermine. Carnation SAMDC genes have an upstream open reading frame (uORF) of 52 or 54 amino acids in 5¢-leader sequences with an extensive stem-loop structure (¡âG > -65 kcal/mol, mfold). SAMDC uORF sequence induced a real protein of 5.8-kDa, which was provided the direct evidence of peptide synthesis from the SAMDC uORF using an in vitro translation system. When we also measured mRNA expression in those mutations, the steady-state mRNA levels were changed. These results suggested that whether stability or inducibility of mRNA is responsible for uORF inhibition. Secondary structure of uORF was strictly required for repression of downstream main ORF and putative phosphorylation sites for protein kinase C, cAMP and cGMP dependent kinase and casein kinase ¥± in uORF might be important for inhibitory effect of uORF. To test whether the uORF protein is really phosphorylated, we performed in vitro phosphoryation assay. In this paper we show the uORF protein specifically is phosphorylated by unknown kinase. It was first reported that phosphorylation of uORF functions as a crucial effect for downstream ORF translation. In addition, the nuclear localization of uORF protein will be examined by constructing the uORF-GFP fusion protein, the expression of which is controlled by the cauliflower mosaic virus 35S promoter. In order to elucidate action mechanism of SAMDC uORF, we have used a primer extension inhibition assay to determine the location and stability of translating ribosomes on the SAMDC gene expression. Our results might imply the distinct mechanism of SAMDC uORF, which was regulated by ribosomal stalling and reinitiation.
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