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Molecular and Cellular Biology, September 2000, p. 6212-6223, Vol. 20, No. 17
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Termination and Peptide Release at the Upstream Open Reading Frame Are Required for Downstream Translation on Synthetic Shunt-Competent mRNA Leaders

Maja Hemmings-Mieszczak,1,* Thomas Hohn,1 and Thomas Preiss2

Friedrich Miescher Institute, CH-4058 Basel, Switzerland,1 and Gene Expression Program, EMBL, D-69117 Heidelberg, Germany2

Received 1 March 2000/Returned for modification 17 April 2000/Accepted 30 May 2000

We have shown recently that a stable hairpin preceded by a short upstream open reading frame (uORF) promotes nonlinear ribosome migration or ribosome shunt on a synthetic mRNA leader (M. Hemmings-Mieszczak and T. Hohn, RNA 5:1149-1157, 1999). We have now used the model mRNA leader to study further the mechanism of shunting in vivo and in vitro. We show that a full cycle of translation of the uORF, including initiation, elongation, and termination, is a precondition for the ribosome shunt across the stem structure to initiate translation downstream. Specifically, AUG recognition and the proper release of the nascent peptide are necessary and sufficient for shunting. Furthermore, the stop codon context must not impede downstream reinitiation. Translation of the main ORF was inhibited by replacement of the uORF by coding sequences repressing reinitiation but stimulated by the presence of the virus-specific translational transactivator of reinitiation (cauliflower mosaic virus pVI). Our results indicate reinitiation as the mechanism of translation initiation on the synthetic shunt-competent mRNA leader and suggest that uORF-dependent shunting is more prevalent than previously anticipated. Within the above constraints, uORF-dependent shunting is quite tolerant of uORF and stem sequences and operates in systems as diverse as plants and fungi.

* Corresponding author. Mailing address: Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4058 Basel, Switzerland. Phone: 41 61 6976684. Fax: 41 61 6973976. E-mail: mieszcza{at}fmi.ch.

Molecular and Cellular Biology, September 2000, p. 6212-6223, Vol. 20, No. 17
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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