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. 2018 Jul 27;46(13):6841-6856.
doi: 10.1093/nar/gky327.

Structural and Kinetic Insights Into Stimulation of RppH-dependent RNA Degradation by the Metabolic Enzyme DapF

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Free PMC article

Structural and Kinetic Insights Into Stimulation of RppH-dependent RNA Degradation by the Metabolic Enzyme DapF

Ang Gao et al. Nucleic Acids Res. .
Free PMC article

Abstract

Vitally important for controlling gene expression in eukaryotes and prokaryotes, the deprotection of mRNA 5' termini is governed by enzymes whose activity is modulated by interactions with ancillary factors. In Escherichia coli, 5'-end-dependent mRNA degradation begins with the generation of monophosphorylated 5' termini by the RNA pyrophosphohydrolase RppH, which can be stimulated by DapF, a diaminopimelate epimerase involved in amino acid and cell wall biosynthesis. We have determined crystal structures of RppH-DapF complexes and measured rates of RNA deprotection. These studies show that DapF potentiates RppH activity in two ways, depending on the nature of the substrate. Its stimulatory effect on the reactivity of diphosphorylated RNAs, the predominant natural substrates of RppH, requires a substrate long enough to reach DapF in the complex, while the enhanced reactivity of triphosphorylated RNAs appears to involve DapF-induced changes in RppH itself and likewise increases with substrate length. This study provides a basis for understanding the intricate relationship between cellular metabolism and mRNA decay and reveals striking parallels with the stimulation of decapping activity in eukaryotes.

Figures

Figure 1.
Figure 1.
Parallels between 5′-end-dependent mRNA degradation pathways in eukaryotes and E. coli. In eukaryotes, cap removal by mRNA-decapping enzyme subunit 2 (Dcp2) bound to Dcp1 triggers mRNA decay (1). The activity of Dcp2 is modulated by several protein factors, including Enhancer of mRNA Decapping (Edc1) (3). The decapped RNA is then degraded by 5′-3′ exoribonuclease 1 (XRN1). In E. coli, the corresponding pathway begins with stepwise removal of the γ and β phosphates of a triphosphorylated transcript by the consecutive action of an unidentified enzyme and RppH (6). RppH activity is stimulated by its interaction with the metabolic enzyme DapF. The resulting monophosphorylated RNA 5′ end potentiates internal cleavage by the endonuclease RNase E.
Figure 2.
Figure 2.
Crystal structure of the E. coli RppH–DapF complex in the apo form with 2:2 stoichiometry. Cartoon representation of the structure as viewed from the front (A) and side (B). The structure comprises two 1:1 RppH–DapF complexes related by 2-fold symmetry in the crystal.
Figure 3.
Figure 3.
Crystal structure of an RppHt-DapFm heterodimer bound to ppcpAGU RNA. (A) Overall view. The proteins are in cartoon representation, and the RNA ligand is in stick representation. RNA is colored violet, with nitrogen, oxygen and phosphorus atoms shown in their ‘atomic’ colors (blue, red and yellow, respectively). Mg2+ cations are depicted by light pink spheres. (B) Surface representation of the proteins, highlighting the shape complementarity of the RppH–DapF interface. (C) All-atom superposition of the RppHt-DapFm-RNA complex with isolated E. coli DapF and RppH-RNA structures (shown in grey color).
Figure 4.
Figure 4.
The RppH–DapF interface. (A) Interface between RppHt (green) and DapFm (cyan). The locations of several amino acids defining the boundaries of the interacting regions are indicated. (B) Contacts between RppH and DapF. Amino acids located ≤3.5 Å from atoms of the partner protein are shown in sticks. Potential hydrogen bonds and cation-π interactions are represented by dashed black and red lines, respectively. (C) View highlighting intermolecular interactions that involve W130, R134 and R145 of RppH. (DF) Conservation of the amino acids shown in panel (B). (D) and (F) show the interacting surfaces of RppH and DapF, respectively. (E) depicts a side view of the RppH–DapF complex. Identical and similar residues conserved in >70% (orange), 50–69% (yellow), or <50% (green for RppH and cyan for DapF) of a representative set of protein sequences from γ- and β-proteobacteria (43) are indicated. Residues colored grey are >3.5 Å away from the partner protein. Panel (F) highlights the position of W130, R134 and R145 (in sticks) of RppH near conserved interface residues of DapF.
Figure 5.
Figure 5.
Heteromeric binding affinity of RppH and DapF. (A) Size exclusion chromatography (SEC) of the RppH–DapF complex (black line), RppH (red line) and wild type DapF (blue line). RppH was added in excess to DapF during complex formation. The RppH–DapF peak at 70 ml corresponds to a complex with 2:2 stoichiometry. (B) SEC of a mixture of RppHW130A,R145A and DapF. W, R correspond to W130A and R145A mutations in RppH. (C) Binding affinity of RppH–DapF complexes, as determined by bio-layer interferometry (BLI). KD values are averages of two experiments in which the concentration of the protein in solution was varied. SE, standard error. ‘Surface’ and ‘solution’ identify the immobilized protein and the protein in solution, respectively. V, F and L correspond to V19S, F58S and L89S mutations in DapF, W and R to W130A and R145A mutations in RppH, and R* to R134A in RppH, respectively. (DG) Association and dissociation curves for representative BLI experiments with RppH and DapF. (D), RppH and DapF; (E), DapF and RppHW130A,R145A; (F), RppH and DapF V19S,F58S,L89S; (G), RppHR134A and DapF. The first protein named in each panel title was immobilized. Concentrations of the protein in solution were 5 nM (violet), 10 nM (blue), 20 nM (cyan), 39 nM (green), 79 nM (yellow), 156 nM (orange) and 312.5 nM (red) in (D), (F) and (G). In (E), the concentrations were 0.35 μM (violet), 0.7 μM (blue), 1.4 μM (cyan), 2.8 μM (green), 5.6 μM (yellow), 11.25 μM (orange) and 22.5 μM (red).
Figure 6.
Figure 6.
Identification of potential allosteric interactions of DapF with RppH. (A) Effect of DapF on RppH reactivity with ppAGU and pppAGU RNA substrates, as analyzed by chromatography. pppAGU substrate: RppH alone, black lines and circles; RppH with DapF, red lines and triangles. ppAGU substrate: RppH alone, blue lines and inverted triangles; RppH with DapF, orange lines and diamonds. Error bars represent standard deviations. Inset: the straight lines obtained in semilogarithmic plots show that the reaction of the triphosphorylated substrate proceeded with first-order kinetics. The rate constants with pppAGU were 0.080 ± 0.001 min−1 for RppH and 0.165 ± 0.014 min−1 for the RppH and DapF mixture (average ± SD, n = 2). (B) All-atom superposition of the RppHt–ppcpAGU–DapFm (protein in green, RNA and Mg2+ cations in violet) and RppHt–2Mg–ppcpAGU (orange) structures. Several RNA- and Mg2+-binding residues are shown in sticks. Blue arrows show conformational shifts in the upper part of the catalytic and Gua-2 binding sites. (C) All-atom superposition of the RppH–DapF (green) and RppH (orange) structures. RNA- and Mg2+-binding residues that adopt different conformations in the two structures are shown in sticks. (D and E) Zoomed-in views of the structure superposition (panel B), centered on the Gua2 binding cleft (D) and the active site (E). All residues are shown in lines except residues participating in RNA and Mg2+ recognition, which are in sticks. (F) Zoomed-in view of the structure superposition (panel C), centered on the Gua2 binding cleft and the upper part of the active site. (G) Schematics of RNA 5′-end recognition in different RppH complexes. The left and central panels are for structures from (13), and the right panel is from the current study. Magenta circles, magnesium ions; green circles, phosphates; dashed white circle, structurally disordered γ phosphate; arrow, location of a water molecule poised for nucleophilic attack.
Figure 7.
Figure 7.
DapF stimulation of RppH reactivity with diphosphorylated RNA substrates. (A) Variable effect of DapF on RppH reactivity with diphosphorylated RNA substrates of different lengths. Phosphate release was quantified by a colorimetric molybdate assay. RppH alone, black lines and circles; RppH complexed with DapF, red lines and triangles; mixture of RppH and DapF V19S,F58S,L89S, blue lines and inverted triangles. The RNA substrates were: ppAGU (3 nt), ppAGUUU (5 nt), ppAGUUUU (6 nt), ppAGUUUUU (7 nt), ppAGUUUUUU (8 nt), and ppAGUUUUUUG (9 nt). Error bars represent standard deviations. (B) Rate constants of the experiments in (A). Each value is the average of at least two experiments (n = 2). SD, standard deviations. (C) Effect of monomeric DapFm on the reactivity of RppH with the 8-nt diphosphorylated substrate (in purple). A control reaction with wild-type DapF is in red.
Figure 8.
Figure 8.
DapF stimulation of RppH reactivity with triphosphorylated RNA substrates. (A) Variable effect of DapF on RppH reactivity with triphosphorylated RNA substrates of different lengths. Pyrophosphate release was quantified by a colorimetric assay. RppH alone, black lines and circles; RppH complexed with DapF, red lines and circles. The RNA substrates were: pppAGU (3 nt), pppAGUUUU (6 nt), pppAGUUUUU (7 nt), and pppAGUUUUUUG (9 nt). Error bars represent standard deviations. Insets: the same data plotted semilogarithmically. (B) Comparison of the reaction time courses for RppH in the presence of DapF, as shown in (A). 3-, 6-, 7- and 9-nt RNAs are shown in black, red, blue and purple colors, respectively. (C) Rate constants of the reactions in (A). SD, standard deviations, n = 2.
Figure 9.
Figure 9.
Effect of DapF on the reactivity of RppH with yeiP mRNA. (A) Influence of DapF on the reaction of RppH with yeiP-U2G mRNA in vitro. Diphosphorylated (DiP) or triphosphorylated (TriP) mRNA that had been synthesized by in vitro transcription was treated for 30 s with purified RppH in the absence or presence of DapF, and the production of monophosphorylated yeiP-U2G was subsequently detected by PABLO. M, PABLO ligation yield of fully monophosphorylated mRNA. Almost all of the monophosphorylated reaction product underwent ligation. The small amount of ligation product observed before treatment with RppH was due to trace ligation of the diphosphorylated starting material (6). Images for the reactions in the presence of DapF are reproduced from portions of Fig. 1 in (6). The second nucleotide of the yeiP transcript was changed from U to G to enable synthesis by T7 RNA polymerase. (B) Decay of yeiP mRNA in E. coli containing wild-type DapF or the DapFV19S,F58S,L89S (VFL) mutant. (Top) Northern blots. Log-phase cultures of isogenic E. coli strains bearing either a wild-type (WT) or mutant (VFL) dapF allele were treated with rifampicin to arrest transcription, and equal amounts of total RNA isolated at time intervals thereafter were analyzed by gel electrophoresis and blotting to detect yeiP mRNA. (Bottom) Semilogarithmic plots of the intensity of the yeiP band as a function of time. Data from a representative experiment are shown. The half-life of yeiP mRNA increased from 1.9 ± 0.1 min in wild-type DapF cells (WT) to 3.7 ± 0.2 min in isogenic DapFV19S,F58S,L89S cells (VFL).
Figure 10.
Figure 10.
Parallels between 5′-terminal RNA deprotection complexes in bacteria and eukaryotes. (A) Superposition of E. coli RppHt–ppcpAGU–DapFm (green, violet and cyan, respectively) and yeast Dcp2–Dcp1–Edc1 (40) (orange, pink, and hot pink, respectively) structures on the Nudix domains. The Box B helix of Dcp2 is indicated. (BC) Views of protein surfaces in electrostatic potential representation for RppH–RNA–DapF (B) and Dcp2–Dcp1–Edc1 (C) complexes, highlighting RNA-binding regions and putative paths (violet lines) of a long RNA ligand. RNA from the RppH–RNA–DapF complex is projected into the Dcp2–Dcp1–Edc1 structure for orientation. Two 5′-terminal RNA nucleotides bound to the catalytic center and nucleobase-binding site of RppH are shown in violet sticks, and the positively charged surface patches mentioned in the text (A, B and C) are encircled by yellow dashed lines. (D) Formation of the RppH–DapF complex. The proteins are separated in orientations favorable for binding. (E) RppH–DapF interface. DapF, whose interface residues are depicted in cyan sticks, partially blocks positively-charged patch C on RppH, which is depicted in surface representation.

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