Bulged-out nucleotides protect an antisense RNA from RNAse III cleavage

Abstract

Bulged-out nucleotides or Internal loops are present In the stem-loop structures of several antisense RNAs. We have used the antisense/target RNA system (CopA/ CopT) that controls the copy number of plasmid R1 to examine the possible biological function of bulged-out nucleotides. Two regions within the major stem-loop of the antisense RNA., CopA, carry bulged-out nucleo tides. Base pairing in either one or both of these regions of the stem was restored by site-specific mutagenesis and in one case a new internal loop was Introduced. The set of mutant and wild-type CopA variants was char acterized structurally in vitro. The resufts reported here indicate a possible function of the bulges: their pres ence protects CopA RNA from being a substrate for the double-strand-specific enzyme RNase III. in vitro cleav age rates were drastically increased when either the lower or both bulges were absent. This Is paralleled by a similar, but not identical, effect of the bulges on metabolic stability of the CopA RNAs in vitro. The degradation pathways of wild-type and mutant CopA In various strain backgrounds are discussed. in the accompanying paper, we address the significance of bulges In CopA for binding to the target RNA in vitro and for its inhibitory efficiency in vitro © 1995 Oxford University Press.