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Biochemical and in silico identification of the active site and the catalytic mechanism of the circadian deadenylase HESPERIN

FEBS Open Bio. 2020; 
Rafailia A A Beta, Athanasios Kyritsis, Veroniki Douka, Eirini Papanastasi, Marianna Rizouli, Demetres D Leonidas, Dimitrios Vlachakis, Nikolaos A A Balatsos
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Molecular Biology Reagents … blocking buffer (1:25000) and visualisation was performed with LumiSensor chemiluminescent HRP substrate kit (GenScript, USA). Homology Modelling … Proceedings of the National Academy of Sciences of the United States of America 117, 993-999 17 … Get A Quote

摘要

The 24 h molecular clock is based on the stability of rhythmically expressed transcripts. The shortening of the poly(A) tail of mRNAs is often the first and rate-limiting step that determines the life-span of a mRNA and is catalyzed by deadenylases. Herein, we determine the catalytic site of Hesperin, a recently described circadian deadenylase in plants, using a modified site-directed mutagenesis protocol and a custom vector, pATHRA. To explore the catalytic efficiency of AtHESPERIN we investigated the effect of AMP and neomycin, and utilized molecular modelling simulations to propose a catalytic mechanism. Collectively, the biochemical and in silico results classify AtHESPERIN in the EEP deadenylase superfamil... More

关键词

AtHESPERIN, Circadian rhythms, Deadenylation, mRNA decay, poly(A) tail