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J. Biochem, 2003, Vol. 133, No. 4 485-491
© 2003 Japanese Biochemical Society

MOLECULAR BIOLOGY

A Novel Screening System for Self-mRNA Targeting Proteins

Bei-Wen Ying1, Tsutomu Suzuki1, Yoshihiro Shimizu2 and Takuya Ueda+,1

1 Department of Integrated Biosciences, Graduate School of Frontier Sciences, and 2 Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, FSB-401, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562

Here we describe the application of an in vitro translation system for genetic screening, to identify RNA-binding proteins that bind to their own mRNAs. It is a relatively novel system designed using an advanced cell-free translation system reconstructed with purified translational components. Due to the absence of nucleases and proteases, the complex of mRNA and nascent polypeptide synthesized in this system is expected to exhibit high stability ensuring the following efficient selection toward the protein. Escherichia coli ribosomal protein S15, which is known to bind to its own mRNA, was employed as a model molecule to evaluate the system. Wild-type S15 mRNA specifically isolated from a mutant mRNA lacking the secondary structure responsible for binding the S15 protein accumulated markedly after several rounds of selection–amplification. The success of this selection demonstrates the potentiality of the systematic screening of self-mRNA targeting proteins through direct and functional selection. This strategy as a method to identify peptides or proteins that bind to their own mRNAs, is of general interest and has different potential applications, such as, the identification of new regulatory proteins or peptide motifs for RNA recognition, the study of self-mRNA–protein interactions, etc.

+ To whom correspondence should be addressed. Tel: +81-471-36-3641, Fax: +81-471-36-3642, E-mail: ueda{at}k.u-tokyo.ac.jp


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