Efficient hammerhead ribozyme and antisense RNA targeting in a slow ribosome Escherichia coli mutant.
We have evaluated inhibition of the plasmid-born chloramphenicol acetyl transferase gene (CAT) by the hammerhead ribozyme and antisense RNA in Escherichia coli where the translation and transcription rates have been modified. Whereas neither antisense nor the hammerhead had an inhibitory effect on CAT activity in wild-type E. coli, both reduced the level of the messenger RNA and the activity of the CAT gene by almost 60% in a slow ribosome mutant. Streptomycin, which increases the speed of translation in this mutant strain, restored full CAT activity. The level of CAT activity expressed from a T7 RNA polymerase promoter was not affected by the presence of either antisense RNA or the hammerhead ribozyme. When the target gene was expressed from a chromosomal locus in wild-type E. coli, both antisense RNA and the hammerhead ribozyme showed some inhibitory activity, but the level of inhibition was significantly increased in the slow ribosome strain. This bacterial system offers a unique entry to the study of cellular factors which mediate the activity of ribozymes in vivo.
Chen
H
H
Département de Biochimie, Université de Montréal, Québec, Canada.
Ferbeyre
G
G
Cedergren
R
R
D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D018014:Gene Transfer Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D016372:RNA, Antisense / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D012270:Ribosomes / Q000378:metabolism*; D013307:Streptomycin / Q000494:pharmacology; D014764:Viral Proteins
D009838:Oligodeoxyribonucleotides; D016372:RNA, Antisense; D016337:RNA, Catalytic; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D015500:Chloramphenicol O-Acetyltransferase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D013307:Streptomycin