RNase III: Genetics and function; structure and mechanism

Abstract

RNase III is a global regulator of gene expression in Escherichia coli that is instrumental in the maturation of ribosomal and other structural RNAs. We examine here how RNase III itself is regulated in response to growth and other environmental changes encountered by the cell and how, by binding or processing double-stranded RNA (dsRNA) intermediates, RNase III controls the expression of genes. Recent insight into the mechanism of dsRNA binding and processing, gained from structural studies of RNase III, is reviewed. Structural studies also reveal new cleavage sites in the enzyme that can generate longer 3' overhangs.

Figure 1

Representative RNase III proteins and RNA oligos used and/or observed in the structures of Aquifex aeolicus (Aa) RNase III-RNA complexes. (a) Homo sapiens Dicer (1,922 amino acid residues, UniProtKB Q9UPY3), Giardia intestinalis Dicer (754 residues, UniProtKB A8BQJ3), H. sapiens Drosha (1,374 residues, UniProtKB Q9NRR4), Trypanosoma brucei mRPN1 (486 residues, UniProtKB Q384H9), Kluyveromyces polysporus Dcr1 (558 residues, UniProtKB A7TR32), Candida albicans Dcr1 (611 residues, UniProtKB Q5A694), Saccharomyces cerevisiae Rnt1p (471 residues, UniProtKB Q02555), Escherichia coli RNase III (226 residues, UniProtKB P0A7Y0), and Bacillus subtilis Mini-III (143 residues, UniProtKB O31418). The scale on top indicates the lengths of polypeptide chains. Domains are indicated by colored boxes. The red box in the RNase III domain indicates the RNase III signature motif. (b) Small synthetic RNAs used in this and previous structural analyses as referenced, namely RNA1 (13); RNA2, RNA3, and RNA4 (42); RNA5 and RNA6 (43); RNA7, RNA8, and RNA9 (41); RNA10, RNA11, and RNA12 (this work). Red arrowheads indicate sites where cleavage occurred during crystallization.

Figure 2

The type II CRISPR (clustered regularly interspersed short palindromic repeats) pathway is outlined from the CRISPR operonic DNA to its guide RNA product. Tracr-RNA is a trans-encoded sRNA transcribed independently of the CRISPR operon. This CRISPR system uses RNase III to process the preCRISPR RNA and was first found in Streptococcus pyogenes.

Figure 3

The RNA polymerase/λ N transcription antitermination complex transcribing the N gene (a) before or (b) after cleavage at the RNaselll-sensitive hairpin (RTS). The RNA transcript in black is transcribed from the P_L promoter and contains the N/Nus factor-binding site (NUTL), the RNase III sensitive hairpin (RTS), the ribosome-binding site (SD). The scissors represent cleavage by RNase III. Ribosome subunits 30S and 50S are indicated.

Figure 4

Sequence and structure of bacterial RNase IIIs. (a) Structure-based sequence alignment of AaRNase III (UniProtKB O67082), TmRNase III (UniProtKB Q9X0I6), and EcRNase III (UniProtKB P0A7Y0). The RNase III domain (RIIID) and double-stranded RNA-binding domain (dsRBD) are boxed. Secondary structural elements are shaded (helices in gray and strands in yellow). Underlined in red are the RNase III signature motif and the linker between RIIID and dsRBD. RNA-binding motifs (RBMs) 1–4 are underlined in blue. (b) The location of RBMs 1–4 in the AaRNase III-Mg²⁺-RNA9 structure (PDB entry 2NUG). One subunit of AaRNase III (backbone in cyan) is shown with the RNA pseudoduplex (molecular surface in light gray). The cleavage site is indicated by the position of two Mg²⁺ ions (spheres in black). Highlighted are the RBMs blue) and the linker between the RIIID and dsRBD (red). (c) Schematic illustration of AaRNase III-RNA interactions as observed in the AaRNase III-Mg²⁺- RNA9 structure (PDB entry 2NUG), showing two subunits of AaRNase III (blue and red), each containing a RIIID (RBMs 3–4) and a dsRBD (RBMs 1–2). The nucleotide residues in the dsRNA are shown as small rectangle boxes, and the two cleavage sites (CSs 1–2) are indicated with arrowheads. The conserved amino acid residue H27 from subunit 1 interacts with the 11th nucleotide residue from CS2, dictating the typical product length of 11 nts when bacterial RNase III processes long dsRNA. The proximal, middle, and distal boxes are outlined and indicated with P, M, and D. The nucleotide residue in the cleavage site is numbered R 0; the rest are numbered according to the polarity of the RNA strand.

Figure 5

Protein fold, RNase III-domain (RIIID) dimerization, catalytic assembly, and cleavage mechanism. (a) Schematic view of the AaRNase III-Mg²⁺-RNA9 structure (PDB entry 2NUG). The two RIIIDs are shown as molecular surfaces, the two double-stranded RNA-binding domains (dsRBDs) are illustrated as ribbon diagrams (α-helices as spirals, β-strands as arrows, and loops as pipes), and the two subunits are colored in cyan and orange. The Mg²⁺ ions are shown as black spheres, and the two RNA strands are shown as tube-and-stick models in blue and red. (b) In the AaRNase III-Mg²⁺-RNA9 structure (PDB entry 2NUG), each cleavage site assembly is composed of the 3′ hydroxyl and 5′ phosphoryl products, two Mg²⁺ ions, three water molecules, and four catalytic side chains; the assemblyexhibits alternate conformations at the ratio of 0.3/0.7 (41). In the minor conformation (30% probability), the distance between the 3′ hydroxyl and the 5′ phosphorus is 3.0 A, shorter than the sum of the van der Waals radii of phosphorus (1.9 A) and oxygen (1.4 A), thus representing the cleavage site arrangement immediately after RNA cleavage. The amino acid and nucleotide residues are shown as stick models and Mg²⁺ ions, and water oxygens are shown as spheres in atomic color scheme (C, gray; N, blue; O, red; P, orange; Mg, black). Metal coordination bonds are indicated by solid lines, and hydrogen bonds are indicated by dashed lines. The nucleotide residue in the middle is numbered R 0, and the rest are numbered according to the polarity of the RNA strand. (c) The stepwise model for phosphoryl transfer and product release. The molecular models for the protein-substrate complex (substrate) and protein-reaction intermediate complex (intermediate) are constructed on the basis of the cleavage-site arrangement immediately after the RNA cleavage (product-0; minor conformation in PDB entry 2NUG), whereas the release of the 5′-end product (product-1) followed by the 3′-end product (product-2) from the cleavage site is represented by the major conformation in the AaRNase III-Mg²⁺-RNA9 structure (PDB entry 2NUG) and the AaRNase III-Mg²⁺-RNA8 structure (PDB entry 2NUF). Panel c was originally published in Reference 41.

Figure 6

Cooperative binding mode of AaRNase III to dsRNA. (a) The cooperative binding mode of AaRNase III to dsRNA as observed in the AaRNase III-Mg²⁺-RNA9 structure (PDB entry 2NUG). The AaRNase III is illustrated as a graphic model with the two subunits colored in pale cyan and light orange, and each subunit is outlined by a transparent molecular surface. The pseudoduplex RNA is illustrated as a tube-and-stick model in gray with the product siRNA highlighted in blue and red. (b) Schematic illustration showing how the siRNA length is determined by the distance between the processing centers of adjacent RIIID dimers.

Figure 7

Hypothetical pathways leading to two functional forms of RNase III. Six distinct conformational states are represented by (a) RNA-free TmRNase III (PDB entry 1O0W), (b) AaRNase IIIE^110Q-RNA2 (PDB entry 1YZ9), (c) AaRNase III-RNA3 (PDB entry 1YYW), (d) AaRNase III^D44N-Mg²⁺-RNA6 (PDB entry 2EZ6), (e) AaRNase III-RNA4 (PDB entry 1YYO), and (f) AaRNase III^E110K-RNA1 (PDB entry 1RC7). The RNase III domain (RIIID) dimer is illustrated as a molecular surface with positive and negative potentials indicated by blue and red, respectively; the double-stranded RNA-binding domains (dsRBDs) are shown as backbone worms in white; and the dsRNA is represented as stick models in atomic color scheme (carbon in white, nitrogen in blue, oxygen in red, phosphorous in yellow). The direction of predicted rotation of the dsRBD-dsRNA complex, enabled by the flexible linker between the RIIID and dsRBD, is indicated with arrowheads on the circles. The orientation of the RIIID moiety was kept constant. Panels a, b, c, e, and f were originally published in Reference 42, whereas panel d is new.

Figure 8

The type II RNA cleavage event. (a) The RNase III-dsRNA interactions observed in the AaRNase III-Mg²⁺-RNA10-CMP and AaRNase III-Mg²⁺-RNA12-AMP structures are schematically illustrated. The dimeric protein is shown as two rectangles and labeled as subunit 1 and subunit 2, and the four RNA-binding motifs (RBMs) as ellipses. The cleavage sites (CSs 1–4) are indicated with arrowheads, and the proximal, middle, and distal boxes are outlined and indicated with one letter abbreviations. For each RNA strand, the nucleotide residue between the two cleavage sites is numbered R 0, and the rest are numbered according to the polarity of the RNA strand. (b) The creation of a 2-, 3-, or 4-nt 3′ overhangs by type I, type I followed by type II, or type II cleavage events, respectively. The type I (CS1, CS2) event is commonly seen in vivo and in structures. The type I followed by typeII event (CS1, CS2; CS3, CS4) is first described here in the two structures above yielding 3-nt overhangs. The type II only event (CS3, CS4) has only been seen in vivo and yields 4-nt overhangs (4). The other type I followed by type II events (CS1, CS4 and CS2, CS3) are hypothetical and have not been seen.