Antisense RNA: Effect of ribosome binding sites, target location, size, and concentration on the translation of specific mRNA molecules

A series of plasmids were constructed to generate RNA complementary to the β-galactosidase messenger RNA under control of the phage lambda P_L promoter. These plasmids generate anti-lacZ mRNA bearing or lacking a synthetic ribosome binding site adjacent to the lambda P_L promoter and/or the lacZ ribosome binding site in reverse orientation. Fragments of lacZ DNA from the 5′ and/or the 3′ region were used in these constructions. When these anti-mRNA molecules were produced in Escherichia coli 294, maximal inhibition of β-galactosidase synthesis occured when a functional ribosome binding site was present near the 5′ end of the anti-mRNA and the anti-mRNA synthesized was complementary to the region of the mRNA corresponding to the lacZ ribosome binding site and/or the 5′-coding sequence. anti-mRNAs producing maximal inhibition of β-galactosidase synthesis exhibited an anti-lacZ mRNA: normal lacZ mRNA ration of 100:1 or higher. Those showing lower levels of inhibition exhibited much lower anti-lacZ mRNA: normal lacZ mRNA ratios. A functional ribosome binding site at the 5′-end was found to decrease the decay rate of the anti-lacZ mRNAs. In addition, the incorppration of a transcription terminator just downstream of the antisense segment provided for more efficient inhibition of lacZ mRNA translation due to synthesis of smaller and more abundant anti-lacZ mRNAs. The optimal constructions produced undetectable levels of β-galactosidase synthesis.