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Nucleic Acids Research, 1989, Vol. 17, No. 7 2463-2476
© 1989

MOLECULAR BIOLOGY

Modulations of the in vitro translational efficiencies of Yellow Fever virus mRNAs: interactions between coding and noncoding regions

Andrès Ruiz-Linares+, Michèle Bouloy, Marc Girard and Annie Cahour*

Unit of Molecular Virology CNRS UA 545, Institut Pasteur, 75724 Peris Cedex 15, France +Present address: Department of Genetics, University of Cambridge Cambridge CB2 3EH, UK

*To whom correspondence should be addressed

Received December 8, 1988. Revised February 23, 1989. Accepted February 23, 1989.

As an approach to define the structural features within the 5' noncoding region of Yellow Fever virus (YFV) that modulate mRNA translational efficiency, we have studied how minor changes In this region affect the translational capacity in vitro of the corresponding mRNAs. A cDNA sequence coding for part of the YFV structural proteins was inserted into the vector pGEM3 containing the bacteriophage T7 promoter. This vector was engineered by site-directed mutagenesis to permit in vitro synthesis of transcripts containing only 5 vector nucleotides at their 5' end. The sequence of the YFV 5' untranslated region was further modified in order to alter the secondary structure of resulting T7 transcripts. The efficiency of these messengers in programing cell-free translation systems varied from 1- to 15-fold, correlating inversely with the potential of the 5' untranslated sequences to form stable secondary structures. A chimaeric messenger containing the YFV 5' noncoding (5' NC) region lmked to a heterologous mRNA derived from Germiston virus, was tested for its in vitro translatability. We found a translational efficiency about 2-fold higher than that obtained with homologous transcripts, suggesting that YFV 5' NC region can function as a potential enhancer for gene expression. Data obtained with a series of plasmids constructed by linking the native YFV 5'NC region to various coding regions of the YFV genome indicated that interactions between the untranslated sequence and protein coding regions influence mRNAs translational efficiency.


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