Abstract
The RNase III family of endoribonucleases participates in maturation and decay of cellular and viral transcripts by processing of double-stranded RNA. RNase III degradation is inherent to most antisense RNA-regulated gene systems in Escherichia coli. In the hok/sok system from plasmid R1, Sok antisense RNA targets the hok mRNA for RNase III-mediated degradation. An intermediate in the pairing reaction between Sok RNA and hok mRNA forms a three-way junction. A complex between a chimeric antisense RNA and hok mRNA that mimics the three-way junction was cleaved by RNase III both in vivo and in vitro. Footprinting using E117A RNase III binding to partially complementary RNAs showed protection of the 13 base pairs of interstrand duplex and of the bottom part of the transcriptional terminator hairpin of the antisense RNA. This suggests that the 13 base pairs of RNA duplex are coaxially stacked on the antisense RNA terminator stem-loop and that each stem forms a monomer half-site, allowing symmetrical binding of the RNase III dimer. This processing scheme shows an unanticipated diversity in RNase III substrates and may have a more general implication for RNA metabolism.
Highlights
RNase III of Escherichia coli [1] belongs to a family of double strand-specific endoribonucleases [2, 3], which have retained interspecies substrate cleavage specificity [4]
Sok antisense RNA consists of an 11-nucleotide 5Ј-single-stranded tail that is responsible for the initial interaction with hok mRNA and a hairpin that functions as a Rho-independent transcriptional terminator (Fig. 1A)
To test if all 13 bp of the duplex were required for processing, we examined 3Ј-end-shortened hok mRNA fragments of 7 and 10 nucleotides for RNase III-mediated CARNA cleavage (Fig. 4D). hok7 failed to sustain cleavage (Fig. 4D, left panel), whereas partial cleavage was observed with hok10
Summary
(Received for publication, February 22, 1999, and in revised form, June 24, 1999). From the ‡Department of Molecular Biology, Odense University, Campusvej 55, DK-5230 Odense M, Denmark and the §Howard Hughes Medical Institute, Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104. Footprinting using E117A RNase III binding to partially complementary RNAs showed protection of the 13 base pairs of interstrand duplex and of the bottom part of the transcriptional terminator hairpin of the antisense RNA. This suggests that the 13 base pairs of RNA duplex are coaxially stacked on the antisense RNA terminator stem-loop and that each stem forms a monomer half-site, allowing symmetrical binding of the RNase III dimer This processing scheme shows an unanticipated diversity in RNase III substrates and may have a more general implication for RNA metabolism. CA-RNA forms a 13-base pair duplex with hok mRNA, thereby resulting in the generation of a three-way junction (Fig. 1D, left panel) This complex mimics the naturally occurring binding intermediate between hok mRNA and Sok antisense RNA (Fig. 1D, right panel).
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