Double-stranded(ds) RNA has diverse roles in gene expression and regulation, host defense, and genome surveillance in bacterial and eukaryotic cells. A central aspect of dsRNA function is its selective recognition and cleavage by members of the ribonuclease III family of divalent-metal-ion-dependent phosphodiesterases. The processing of dsRNA by RNase III family members is an essential step in the maturation and decay of coding and noncoding RNAs, including miRNAs and siRNAs. RNase III, as first purified from Escherichia coli, has served as a biochemically well-characterized prototype, and other bacterial orthologs provided the first structural information. RNase III family members share a unique fold (RNase III domain) that can dimerize to form a structure that binds dsRNA and cleaves phosphodiesters on each strand, providing the characteristic 2 nt, 3’-overhang product ends. Ongoing studies are uncovering the functions of additional domains, including, inter alia, the dsRNA-binding and PAZ domains that cooperate with the RNase III domain to select target sites, regulate activity, confer processivity, and support the recognition of structurally diverse substrates. RNase III enzymes function in multicomponent assemblies that are regulated by diverse inputs, and at least one RNase III-related polypeptide can function as a non-catalytic, dsRNA-binding protein. This review summarizes the current knowledge of the mechanisms of catalysis and target site selection of RNase III family members, and also addresses less well understood aspects of these enzymes and their interactions with dsRNA.

The maturation and degradation of RNA molecules are essential features of the mechanism of gene expression, and provide the two main points for post-transcriptional regulation. Cells employ a functionally diverse array of nucleases to carry out RNA maturation and turnover. Viruses also employ cellular ribonucleases, or even use their own in their reproductive cycles. Studies on bacterial ribonucleases, and in particular those from Escherichia coli, are providing insight into ribonuclease structure, mechanism, and regulation. Ongoing biochemical and genetic analyses are revealing that many ribonucleases are phylogenetically conserved, and exhibit overlapping functional roles and perhaps common catalytic mechanisms. This article reviews the salient features of bacterial ribonucleases, with a focus on those of E. coli, and in particular, ribonuclease III. RNase III participates in a number of RNA maturation and RNA decay pathways, and is regulated by phosphorylation in the T7 phage-infected cell. Plasmid and phage RNAs, in addition to cellular transcripts, are RNase III targets. RNase III orthologues occur in eukaryotic cells, and play key functional roles. As such, RNase III provides an important model with which to understand mechanisms of RNA maturation, RNA decay, and gene regulation.

The double helix is a ubiquitous feature of RNA molecules and provides a target for nucleases involved in RNA maturation and decay. Escherichia coli ribonuclease III participates in maturation and decay pathways by site-specifically cleaving double-helical structures in cellular and viral RNAs. The site of cleavage can determine RNA functional activity and half-life and is specified in part by local tertiary structure elements such as internal loops. The involvement of base pair sequence in determining cleavage sites is unclear, because RNase III can efficiently degrade polymeric double-stranded RNAs of low sequence complexity. An alignment of RNase III substrates revealed an exclusion of specific Watson-Crick bp sequences at defined positions relative to the cleavage site. Inclusion of these ''disfavored'' sequences in a model substrate strongly inhibited cleavage in vitro by interfering with RNase III binding. Substrate cleavage also was inhibited by a 3-bp sequence from the selenocysteine-accepting tRNA Sec , which acts as an antideterminant of EF-Tu binding to tRNA Sec . The inhibitory bp sequences, together with local tertiary structure, can confer site specificity to cleavage of cellular and viral substrates without constraining the degradative action of RNase III on polymeric double-stranded RNA. Base pair antideterminants also may protect doublehelical elements in other RNA molecules with essential functions.RNA maturation and decay reactions involve site-specific cleavages carried out by a diverse ensemble of cellular ribonucleases. The site of cleavage can determine RNA half-life and functional activity and is established by specific sequence and structural features in RNA (1). Double-helical regions provide a target for enzymatic cleavage and can be formed through long-range or short-range intramolecular base pairing. Double-stranded (ds) RNA structures also are created by antisense RNA binding to complementary RNA sequences, or by symmetric transcription of cellular or viral DNA. Moreover, many viral chromosomes are dsRNA molecules. Thus, dsRNA cleavage reactions are not only necessary for RNA maturation, function, and decay, but also may provide an antiviral strategy (2, 3).Ribonuclease III of Escherichia coli [EC 3.1.24] is a dsRNAspecific nuclease with important functions in cellular and viral RNA metabolism (2, 4, 5). RNase III cleaves the primary transcript of the ribosomal RNA operons to provide the immediate precursors to the mature 16S and 23S ribosomal RNAs. RNase III also participates in the maturation of cellular and phage mRNAs and can control mRNA translation and half-life (2, 5). RNase III is a homodimeric phosphodiesterase, cleaving substrate to create 5Ј-phosphate, 3Ј-hydroxyl termini. The only required cofactor is a divalent metal ion, preferably Mg 2ϩ . Substrate cleavage in vitro at physiologically relevant salt concentrations accurately reflects the in vivo cleavage pattern (2,5,7,8). Nucleases similar to RNase III are present in all cells and perform similar functions, in...

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Ethylation interference and hydroxyl radical footprinting were used to identify substrate ribose‐phosphate backbone sites that interact with the Escherichia coli RNA processing enzyme, ribonuclease III. Two RNase III mutants were employed, which bind substrate in vitro similarly as wild‐type enzyme, but lack detectable phosphodiesterase activity. Specifically, altering glutamic acid at position 117 to lysine or alanine uncouples substrate binding from cleavage. The two substrates examined are based on the bacteriophage T7 R1.1 RNase III processing signal. One substrate, R1.1 RNA, undergoes accurate single cleavage at the canonical site, while a close variant, R1.1[WC‐L] RNA, undergoes coordinate double cleavage. The interference and footprinting patterns for each substrate (i) overlap, (ii) exhibit symmetry and (iii) extend approximately one helical turn in each direction from the RNase III cleavage sites. Divalent metal ions (Mg2+, Ca2+) significantly enhance substrate binding, and confer stronger protection from hydroxyl radicals, but do not significantly affect the interference pattern. The footprinting and interference patterns indicate that (i) RNase III contacts the sugar‐phosphate backbone; (ii) the RNase III‐substrate interaction spans two turns of the A‐form helix; and (iii) divalent metal ion does not play an essential role in binding specificity. These results rationalize the conserved two‐turn helix motif seen in most RNase III processing signals, and which is necessary for optimal processing reactivity. In addition, the specific differences in the footprint and interference patterns of the two substrates suggest why RNase III catalyzes the coordinate double cleavage of R1.1[WC‐L] RNA, and dsRNA in general, while catalyzing only single cleavage of R1.1 RNA and related substrates in which the scissle bond is within an asymmetric internal loop.