Abstract
Ribozymes are potentially powerful tools for the suppression of intracellular gene expression. However, the few reports that exist of their activities in bacteria have described mixed success. Chuat and Galibert (Chuat, J.-C., and Galibert, F. (1989) Biochem. Biophys. Res. Commun. 162, 1025-1029) failed to detect any trans-activities of hammerhead ribozymes in Escherichia coli, while Sioud and Drlica (Sioud, M., and Drlica, K. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 7303-7307) reported complete inhibition of expression of the gene for a nonbacterial protein, HIV-1 integrase, by trans-acting hammerhead ribozymes in E. coli. It is of interest to determine whether ribozymes can really be used in natural bacterial systems (Altman, S. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10898-10900). We now report that a ribozyme designed to cleave the A2 gene of RNA coliphage SP, when transcribed from a plasmid in E. coli caused failure of the proliferation of progeny phage. Inactive ribozymes with altered catalytic sequences did not affect phage growth. These results indicate that it is mainly the catalytic activity of the ribozyme and not its function as an antisense molecule that is responsible for suppressing the proliferation of the RNA phage. Moreover, an analysis based on numbers of plaque-forming units and the function of the A2 protein indicated that antisense RNA may successfully compete with ribosomes in targeting mRNA while ribozymes in this study may not compete with ribosomes in naturally occurring bacterial transcription/translation-coupled systems.
Highlights
Ribozymes are potentially powerful toolsfor the sup. at the CCA end of an oligonucleotide
We report that a ribozyme designed to cleave the A2 gene of RNA coliphage SP, when transcribed from a plasmidin E. coli caused fail
Substrates can have anysequence as long as the cleavage site containsNUX, where N can be any nucleotide and X can be any nucleotide except G, the cleavage occurs most efliciently after the GUC triplet [15, 16].Because of the portability of the hammerhead ribozyme, it has been used as a ure of the proliferation of progeny phage
Summary
AND DISCUSS~ON pressed plaque formation by SP phage (Table I). When these experiments were conducted initially (Experiments 3 and 41, Ribozymes Directedagainst theA2 Gene-The RNA phage SP we had not yet constructed the ANT and INACT-2 control plasencodes a maturation protein ( A 2 ,in Fig. lA),a coat protein mids [19, 27]. It should be pointed out that the plaques consensus sequence was placed in a reversed 5' + 3' orienta- were smaller on the lawns of cells that expressed RIB or tion such that the numbers of each nucleotide remained the ANT thanthat on the lawn of controlcells that carried same as thatof the wild-typeribozyme. These three sequences pJDC408, suggesting that proliferation of phage was suppressed in theformer cells. The abbreviation used is: IPTG,isopropyl-1-thio-8-n-galactopyrano- F'tacIq cells that carried either RIB or ANT was the same as side
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