Expression of the rat interferon-alpha 1 gene in Escherichia coli controlled by the secondary structure of the translation-initiation region

Gene
R A SpanjaardJ van Duin

Abstract

A synthetic ribosome-binding site (RBS) containing a 7-nucleotide-long Shine-Dalgarno (SD) sequence was placed ahead of the rat interferon (IFN)-alpha 1 coding region. The translational efficiency of this construct was extremely low. Structural probing of transcripts with RNases T1 and U2 combined with computer predictions revealed the presence of a stable hairpin in which the SD region was base-paired to codons 3, 4 and 5 of the IFN mRNA. Each mutation in this stem changing an A-U to an A.C or a G-C a G.U pair increased translational efficiency about fourfold and this effect could be reversed by a compensating stabilizing substitution in the other strand of the stem. We conclude that the strength of an RBS is to a major degree determined by its involvement in secondary structure. We also show that the negative effect of secondary structure on the efficiency of an RBS can be overcome by allowing upstream translation to terminate within the base-paired region. In our clones, termination-dependent restarts occur at a frequency comparable to that taking place in constructs containing destabilized hairpins.

References

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Citations

Aug 1, 1995·The International Journal of Biochemistry & Cell Biology·M H de SmitH G van Eijk
Oct 1, 1990·Proceedings of the National Academy of Sciences of the United States of America·M H de Smit, J van Duin
Jul 25, 1989·Nucleic Acids Research·R A Spanjaard, J van Duin
Apr 25, 1993·Nucleic Acids Research·J van HimbergenJ van Duin
Jan 24, 2009·Protein Expression and Purification·Yin GuoViswanath Bandaru
Jan 7, 1994·Journal of Molecular Biology·M H de Smit, J van Duin
Dec 9, 2008·Journal of Molecular Biology·Maarten H de SmitCornelis W A Pleij
Mar 14, 2007·Biochemical and Biophysical Research Communications·Young Seoub ParkGyoo Yeol Jung
Feb 18, 2006·Journal of Molecular Biology·Lisa M JungbauerSilvia Cavagnero

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