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

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.