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 beta-galactosidase messenger RNA under control of the phage lambda PL promoter. These plasmids generate anti-lacZ mRNA bearing or lacking a synthetic ribosome binding site adjacent to the lambda PL 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 beta-galactosidase synthesis occurred 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 5' region of the mRNA corresponding to the lacZ ribosome binding site and/or the 5'-coding sequence. Anti-mRNAs producing maximal inhibition of beta-galactosidase synthesis exhibited an anti-lacZ mRNA:normal lacZ mRNA ratio 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 incorporation 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 beta-galactosidase synthesis.
Daugherty
B L
BL
Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry, Robert Wood Johnson Medical School, Piscataway, NJ 08854-5635.
Hotta
K
K
Kumar
C
C
Ahn
Y H
YH
Zhu
J D
JD
Pestka
S
S
D001665:Binding Sites; D014176:Protein Biosynthesis; D012313:RNA; D016372:RNA, Antisense; D012333:RNA, Messenger / Q000037:antagonists & inhibitors*; D012270:Ribosomes / Q000378:metabolism*; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors / Q000096:biosynthesis
D016372:RNA, Antisense; D012333:RNA, Messenger; D012313:RNA; D001616:beta-Galactosidase