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. 2016 May 6:6:25448.
doi: 10.1038/srep25448.

Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation

Swapna Gone  1 , Mercedes Alfonso-Prieto  2 , Samridhdi Paudyal  3 , Allen W Nicholson  1   3
Affiliations

Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation

Swapna Gone et al. Sci Rep. .

Abstract

Ribonuclease III (RNase III) is a conserved, gene-regulatory bacterial endonuclease that cleaves double-helical structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control, reflective of its global regulatory functions. Escherichia coli (Ec) RNase III catalytic activity is known to increase during bacteriophage T7 infection, reflecting the expression of the phage-encoded protein kinase, T7PK. However, the mechanism of catalytic enhancement is unknown. This study shows that Ec-RNase III is phosphorylated on serine in vitro by purified T7PK, and identifies the targets as Ser33 and Ser34 in the N-terminal catalytic domain. Kinetic experiments reveal a 5-fold increase in kcat and a 1.4-fold decrease in Km following phosphorylation, providing a 7.4-fold increase in catalytic efficiency. Phosphorylation does not change the rate of substrate cleavage under single-turnover conditions, indicating that phosphorylation enhances product release, which also is the rate-limiting step in the steady-state. Molecular dynamics simulations provide a mechanism for facilitated product release, in which the Ser33 phosphomonoester forms a salt bridge with the Arg95 guanidinium group, thereby weakening RNase III engagement of product. The simulations also show why glutamic acid substitution at either serine does not confer enhancement, thus underscoring the specific requirement for a phosphomonoester.

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Figures

Figure 1
Figure 1. Alignment of Escherichia coli (Ec-)RNase III and Aquifex aeolicus (Aa-)RNase III sequences.
Only the catalytic domains (RIIID) of the Ec-RNase III and Aa-RNase III polypeptides are shown. Black highlighted residues indicate conservation and the boxed residues indicate chemical similarity. The secondary structural elements of Aa-RNase III are shown on top. The segment highlighted in red (phospholoop) corresponds to the loop connecting the α 2 and α 3 helices, and contains the serine targets of T7PK (see Results and Discussion). The regions highlighted in blue correspond to the RNA-binding motifs 3 and 4 (RBM3 and RBM4) as described elsewhere. Supplementary Figure S1 provides the alignment of complete RNase III polypeptide sequences.
Figure 2
Figure 2. Alanine mutation identifies Ser33 and Ser34 as in vitro targets of Ec-RNase III phosphorylation by T7PK.
H6-tagged Ec-RIIID polypeptides with the indicated alanine mutations were purified in soluble form and subjected to phosphorylation in vitro using dephosphorylated T7PK and [γ -32P]ATP (see Materials and methods). The concentration of Ec-RIIID (or mutant) or RNase III (or mutant) was 2.5 μ M and dephosphorylated T7PK was 3.3 μ M. Aliquots were electrophoretically fractionated by SDS-PAGE, and removal of unincorporated radioactivity accomplished by gel staining and destaining (see also Materials and methods). Reactions were imaged by phosphorimaging. (a) Effect of single alanine mutations on H6-Ec-RIIID phosphorylation. Positions of (self-phosphorylated) T7PK and RIIID are indicated on the right. The first lane in each gel image displays a control reaction where RIIID was omitted. (b) Effect of double alanine mutations on H6-Ec-RIIID phosphorylation. The upper image is the phosphorimage of phosphorylation reactions involving RIIID with double alanine mutations in the phospholoop. The lower image is the corresponding Coomassie-stained gel image showing the locations of the T7PK and RIIID polypeptides. (c) Effect of double and quadruple alanine mutations on T7PK phosphorylation of Ec-RNase III. S195 and S198 are surface serine residues in the C-terminal dsRBD.
Figure 3
Figure 3. RNase III phosphorylation stimulates R1.1 RNA cleavage in vitro.
(a) Sequence and secondary structure of R1.1 RNA. The positions of protein interaction (pb, proximal box; mb, middle box; and db, distal box) are highlighted with red boxes. The interacting protein domains [RNA-binding motifs (RBMs) 1–4] are indicated in blue. The arrow marks the site of RNase III cleavage. (b) Gel phosphorimage of time-course assays of cleavage of internally-32P-labeled R1.1 RNA (200 nM) by Ec-RNase III (20 nM), in phosphorylated or nonphosphorylated form. Lanes 1–6 shows a representative time course assay involving phosphorylated Ec-RNase III, while lanes 7–12 show the time course assay involving mock-phosphorylated enzyme. Lanes 2–6 and 8–12 show 15 sec, 30 sec, 1 min, 2 min and 4 min reaction time points; lanes 1 and 7 represent control reactions where R1.1 RNA was incubated for 1 min in an otherwise complete reaction, but lacking MgCl2. The RNA doublets at the bottom of the lanes are R1.1 RNA 3′-end-containing products, the longer product of which contains an additional non-templated nucleotide incorporated during R1.1 RNA synthesis. (c) Graphic depiction of time course reactions of R1.1 RNA cleavage by phosphorylated and mock-phosphorylated Ec-RNase III. The points are the average of two experiments, with maximum errors shown. (d) Substrate concentration dependence of the initial rate of cleavage of R1.1 RNA by phosphorylated and mock-phosphorylated Ec-RNase III. Cleavage reactions involved 10 nM Ec-RNase III, and the indicated concentrations of internally 32P-labeled R1.1 RNA. Reactions were performed in duplicate. The kinetic constants are provided in Table 2.
Figure 4
Figure 4. Ec-RNase III homology-modeled structure in complex with cleaved dsRNA.
The protein is represented in gray cartoon form, and the cleaved dsRNA in red. Residues S33, S34, K35 and R95 are shown as licorice with C, O and N atoms in cyan, red and blue, respectively; only polar H atoms (white) are displayed. The two catalytic Mg2+ ions are shown as green spheres, whereas the third Mg2+ ion, which has been proposed to be involved in product release, is in orange. (a) Front view of the structure, along the axis of the cleaved dsRNA. The two subunits of the homodimer are represented in light and dark gray cartoon, respectively. The two domains of homodimeric RNase III (RIIID and dsRBD) are indicated. (b) Side view of the structure, upon 90 degree rotation along the axis perpendicular to the cleaved dsRNA. The location of residues S33, S34, K35 and R95 (and their symmetric counterparts S33′, S34′, K35′ and R95′ in the other subunit of the homodimer) is shown in licorice representation.
Figure 5
Figure 5. Representative interactions of the phosphorylated serine residues.
(a) pS33 and (b) pS34 observed in the molecular dynamics simulations of Ec-RNase III in complex with a minimal size product. Residues S33, S34, K35 and R95 are shown as licorice, with C, O, N and P atoms in cyan, red, blue and ochre, respectively; only polar H atoms (white) are displayed. The remainder of the protein is represented in gray cartoon form.
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References

    1. Arraiano C. M. et al. The critical role of RNA processing and degradation in the control of gene expression. FEMS Microbiol. Rev. 34, 883–923 (2010). - PubMed
    1. Evguenieva-Hackenberg E. & Klug G. New aspects of RNA processing in prokaryotes. Curr. Opin. Microbiol. 14, 587–592 (2011). - PubMed
    1. Rochat T., Bouloc P. & Repoila F. Gene expression control by selective RNA processing and stabilization in bacteria. FEMS Microbiol. Lett. 344, 104–103 (2013). - PubMed
    1. Kime L. et al. The first small-molecule inhibitors of members of the ribonuclease E family. Sci. Rep. 5, 8028 (2015). - PMC - PubMed
    1. Eidem T. M. et al. Small-molecule inhibitors of Staphylococcus aureus RnpA-mediated RNA turnover and tRNA processing. Antimicrob. Agents Chemother. 59, 2016–2028 (2015). - PMC - PubMed

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