Translational suppression by hammerhead ribozymes and inactive variants in S-cerevisiae

The activity of hammerhead ribozymes in S. cerevisiae was assessed using two ribozymes that were designed to intramolecularly attack the hepatitis B viral X mRNA. The ribozymes effectively suppressed the expression of the X-lacZ fusion gene, when they were inserted at the 5' end of the X mRNA. The ribozymes cleaved the target RNA efficiently at the targeted phosphodiester bond, but the inactive mutants carrying GS-to-A substitution in the core did not, as the total RNA preparations of yeast extracts was assayed by primer extension. These G5A mutants, however, exerted the suppression as effectively as the wild-type ribozymes. The results, with several mutations introduced to a ribozyme, suggested that either mere formation of hammerhead-like structures with the three stems, or the formation of any two stems, could inhibit translation. Thus, the hammerhead-like structures, leading to cleavage or not, could effectively suppress translation, especially when formed around the initiation codon. The GS-to-A and U7-to-G mutations and replacement of the stem-II hairpin tetraloop did not appear to affect the formation of the inhibitory structure(s). The inhibition that was observed when stems I and III were directly connected without a loop or with a stem II hairpin was completely when they were connected with only the loop of stem II (not containing the stem portion). Published by Elsevier Science B.V.
Publisher
ELSEVIER SCIENCE BV
Issue Date
2000-06
Language
ENG
Keywords

VIRUS TYPE-1 EXPRESSION; MESSENGER-RNA; GENE-EXPRESSION; MEDIATED CLEAVAGE; ESCHERICHIA-COLI; SELF-CLEAVAGE; HUMAN-CELLS; INHIBITION; ENZYMES; SUBSTRATE

Citation

BIOMOLECULAR ENGINEERING, v.16, no.6, pp.183 - 189

ISSN
1389-0344
DOI
10.1016/S1389-0344(99)00052-0
URI
http://hdl.handle.net/10203/2084
Appears in Collection
BS-Journal Papers(저널논문)
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