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Originally published In Press as doi:10.1074/jbc.M105944200 on August 6, 2001

J. Biol. Chem., Vol. 276, Issue 41, 38036-38043, October 12, 2001
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Polyamine Regulation of Ribosome Pausing at the Upstream Open Reading Frame of S-Adenosylmethionine Decarboxylase*

G. Lynn Law, Alexa Raney, Carrie Heusner, and David R. MorrisDagger

From the Department of Biochemistry, University of Washington, Seattle, Washington 98195-7350

Synthesis of S-adenosylmethionine decarboxylase (AdoMetDC), a key regulated enzyme in the pathway of polyamine biosynthesis, is feedback-controlled at the level of translation by spermidine and spermine. The peptide product of an upstream open reading frame (uORF) in the mRNA is solely responsible for polyamine regulation of AdoMetDC translation. Using a primer extension inhibition assay and in vitro protein synthesis reactions, we found ribosomes paused at or close to the termination codon of the uORF. This pause was greatly diminished with the altered uORFs' sequences that abolish uORF regulation in vivo. The half-life of the ribosome pause was related to the concentration of polyamines present but was unaffected by magnesium concentration. Furthermore, inhibition of translation initiation at a reporter gene placed downstream of the AdoMetDC uORF directly correlated with the stability of the ribosome pause at the uORF. These observations are consistent with a model in which regulation of ribosome pausing at the uORF by polyamines controls ribosome access to the downstream AdoMetDC reading frame.

* This work was supported by National Institutes of Health Research Grant CA39053.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry, University of Washington, Box 357350, Seattle, WA 98195-7350. Tel.: 206-543-1694; Fax: 206-543-4822; E-mail: dmorris@u.Washington.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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