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Nucleic Acids Research, 1992, Vol. 20, No. 10 2503-2510
© 1992

MOLECULAR BIOLOGY

A cell-free extract from yeast cells for studying mRNA turnover

Peter Vreken, Nienke Buddelmeijer and Hendrik A. Raué*

Department of Biochemistry & Molecular Biology, Faculty of Chemistry, Vrije Universiteit de Boelelaan 1083, 1081 HV Amsterdam, The Netherlands

* To whom correspondence should be addressed

Received January 30, 1992. Revised April 15, 1992. Accepted April 15, 1992.

We have isolated a cell-free extract from yeast cells that reproduces the differences observed in vivo in the rate of turnover of individual yeast mRNAs. Detailed analysis of the degradation of yeast phosphoglycerate kinase (PGK) mRNA in this system demonstrated that both natural and synthetically prepared PGK transcripts are degraded by the same pathway previously established by us in vivo, consisting of endonucleolytic cleavage at a number of 5'-GGUG-3' sequence motifs within a short target region located close to the 3'-end of the coding sequence followed by 5'–3' exonucleolytic removal of the resulting fragments. The extract, therefore, is suitable for studying the mechanistic details of mRNA turnover in yeast. As a first application of this system we have performed a limited mutational analysis of two of the GGUG motifs within the endonucleolytic target region of the PGK transcript. The results show that sequence changes in either motif abolish cleavage at the mutated site only, indicating the involvement of the residues in question in selection of the cleavage positions.


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