1. Microbiology and Infectious Disease
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A dedicated diribonucleotidase resolves a key bottleneck for the terminal step of RNA degradation

  1. Soo-Kyoung Kim
  2. Justin D Lormand
  3. Cordelia A Weiss
  4. Karin A Eger
  5. Husan Turdiev
  6. Asan Turdiev
  7. Wade C Winkler  Is a corresponding author
  8. Holger Sondermann  Is a corresponding author
  9. Vincent T Lee  Is a corresponding author
  1. University of Maryland, United States
  2. Cornell University, United States
Research Article
Cite this article as: eLife 2019;8:e46313 doi: 10.7554/eLife.46313
8 figures, 2 tables, 10 data sets and 2 additional files

Figures

Figure 1 with 1 supplement
Orn has a stark preference for diribonucleotide cleavage in vitro.

RNA nucleotides two to seven residues in length (1 μM, containing the corresponding 32P-labeled RNA tracer) were each subjected to cleavage over time with 5 nM (A, B) or 1000 nM OrnVc (C, D). Aliquots of each reaction were stopped at indicated times (min), and assessed by denaturing 20% PAGE (A, C). Quantification of the intensities of bands corresponding to the amount of uncleaved initial oligonucleotide over time are plotted as the average and SD of three independent experiments (B, D).

https://doi.org/10.7554/eLife.46313.003
Figure 1—figure supplement 1
Catalytic residues of OrnVc are required for degradation for 32P-AAAAAGG in vitro.

Degradation of 32P-AAAAAGG by purified indicated His10-MBP-OrnVc variants with 5 nM (A) or 1 µM (B). Samples were stopped at indicated time (min) and analyzed by denaturing 20% PAGE. Representative gel images of two independent assays are shown.

https://doi.org/10.7554/eLife.46313.004
Figure 2 with 2 supplements
Structures reveal Orn’s conserved substrate preference for diribonucleotides.

Crystal structures of pGpG-bound V. cholerae Orn (A) and human REXO2 (B) are shown in comparison to E. coli RNase T bound to substrate (PDB 3nh1; Hsiao et al., 2011) (C), another DnaQ-fold 3’−5’ exoribonuclease with a DEDD(h) active site motif. The top panels show ribbon representations of the dimeric enzymes. The insets are surface representations of the enzymes’ active sites shown in similar orientations. The bottom panel describes the active site residues involved in RNA binding and catalysis. Residue numbering for REXO2 refers to its cytosolic isoform lacking the mitochondria-targeting pre-sequence. The sequence logos in (D) were constructed based on multi-sequence alignments of Orn and RNase T orthologs. Overall sequence identity ranges from 43 to 70% for Orn and 46 to 69% for RNase T. Sequence identifiers are provided in Figure 2—source data 2. Sequence logos were plotted using WebLogo (Crooks et al., 2004). Conserved residues of the active site’s DEDD motif (‘act.’; red), for ribonucleotide base binding (‘nuc.’; gray), and of the phosphate cap (‘P-cap’; purple) are highlighted.

https://doi.org/10.7554/eLife.46313.006
Figure 2—source data 1

Data collection and refinement statistics.

https://doi.org/10.7554/eLife.46313.009
Figure 2—source data 2

Sequences used to calculate surface conservation and generate Weblogos.

https://doi.org/10.7554/eLife.46313.010
Figure 2—figure supplement 1
Structural comparison of Orn orthologs, RNase T and Exo I.

(A) Superposition of OrnVc with REXO2 (left) and the substrate-free state of Orn from Xanthomonas campestris (PDB 2gbz; Chin et al., 2006) (right). Rmsd values are reported for the alignment of a monomer. (B) Surface properties of Orn enzymes. An alignment of Orn orthologs was used to map conservation scores onto the solvent accessible surface of OrnVc (left). High degree of conservation overlaps with the acidic active site observed in the electrostatic potential map calculated with the APBS software (Baker et al., 2001) and based on OrnVc (right). (C) Active-site view of E. coli RNase T bound to dAAC (PDB 3v9z; Hsiao et al., 2012). (D) Active-site view of E. coli Exo I bound to dT13 (PDB 4jrp; Korada et al., 2013).

https://doi.org/10.7554/eLife.46313.007
Figure 2—figure supplement 2
Structural comparison of OrnVc and REXO2 bound to diverse diribonucleotides.

(A) Crystal structures of REXO2 bound to pApA and pApG. The structural overview (left) shows a C-terminal helix that is unique to Orn orthologs in higher eukaryotes and in the current crystal forms is stabilized by crystal packing contacts. (B) Crystal structures of OrnVc bound to di-purine (pApA, pApG, pGpA), purine-pyrimidine (pGpC, pCpG), and di-pyrimidine (pCpU) substrates. Views are identical to those shown in Figure 2 for the REXO2:pGpG and Orn:pGpG complexes.

https://doi.org/10.7554/eLife.46313.008
Figure 3 with 4 supplements
Functional importance of active-site and phosphate-cap residues for Orn function.

(A) In vitro enzyme activity. Degradation of 32P-pGpG (1 μM) by purified wild-type OrnVc or variants with alanine substitutions (5 nM) at the indicated sites was assessed. Samples were stopped at the indicated times (min) and analyzed by denaturing 20% PAGE. Representative gel images are shown with the indicated RNA size. Figure 3—figure supplement 4 shows the graphs of the means and SD of three independent experiments. (B) In vivo activity of alanine substituted ornVc alleles to complement P. aeruginosa Δorn. Overnight cultures of the indicated strains were allowed to stand for 10 min without agitation to allow bacterial aggregates to sediment. Representative images of the cultures of triplicated assays are shown.

https://doi.org/10.7554/eLife.46313.011
Figure 3—figure supplement 1
Contribution of catalytic residues and His10-MBP tag to binding interaction between OrnVc and pGpG.

The dissociation constants (Kd) of indicated purified (A) His10-MBP-OrnVc variants and (B) untagged OrnVc to pGpG were measured by DRaCALA. Fraction bound from data was plotted and determined by nonlinear regression. All data shown represent average and SD of three independent experiments.

https://doi.org/10.7554/eLife.46313.016
Figure 3—figure supplement 2
Immunoblot analysis of Orn in P. aeruginosaorn expressing indicated orn alleles.

Proteins in whole cell lysates of strains expressing the indicated HA-tagged orn allele were separated by 12% SDS-PAGE and probed with anti-HA antibodies. All images were performed in duplicate independently. A representative image of two independent experiments is shown.

https://doi.org/10.7554/eLife.46313.012
Figure 3—figure supplement 3
Molecular weight determination indicates that OrnVc variants with point mutations at the phosphate cap and active site remain dimeric.

(A) Absolute molecular weights (black data points across elution peaks are plotted on the right axis; theoretical monomer and dimer molecular weights, horizontal dashed lines) of wild-type OrnVc and point mutants were determined using SEC-MALS (90°-light scattering: colored, solid lines; refractive index signal: black, dashed lines; plotted on the left axis).

https://doi.org/10.7554/eLife.46313.013
Figure 3—figure supplement 4
pGpG degradation by purified wild-type OrnVc or variants with indicated alanine substitutions.

1 µM pGpG was treated with 5 nM of the indicated Orn protein and sampled at indicated time points. The graphs show quantification of three independent data sets, of which representative radiographs are shown in Figure 3A.

https://doi.org/10.7554/eLife.46313.014
Figure 4 with 1 supplement
OrnVc cleaves all diribonucleotides.

(A) OrnVc (5 nM) was incubated with di-purine (pRpR), purine-pyrimidine (pRpY or pYpR), or di-pyrimidine (pYpY) diribonucleotides (1 μM) containing the corresponding 32P-labeled RNA tracer. Aliquots of each reaction were stopped at the indicated times (min) and assessed by denaturing 20% PAGE. (B) Quantification of the intensities of bands corresponding to the amount of diribonucleotide cleaved at the 10 min time point. Results are the average and SD of duplicate independent experiments. Orn cleaves all diribonucleotides to nucleoside monosphosphates, albeit to varying extents. Diribonucleotides consisting of two purines (pRpR) were hydrolyzed most efficiently, with over 90% of starting RNAs processed by 10 min. A majority (>75%) of diribonucleotides with a 5' purine (pRpY) were also processed by the 10 min endpoint. However, diribonucleotides with a 5' pyrimidines exhibited moderately reduced levels of cleavage. Di-pyrimidine (pYpY) substrates, and in particular pUpC and pCpC, showed the slowest turnover from the substrates tested. These results demonstrate that while all diribonucleotides are acceptable substrates for Orn, the enzyme is likely to exhibit moderate preferences for diribonucleotides that contain a 5' purine.

https://doi.org/10.7554/eLife.46313.017
Figure 4—figure supplement 1
Effect of GpG and pAp on Orn activity.

(A) Cleavage of 130 nM 32P-pGpG by 5 nM Orn in the absence or presence of unlabeled pAp, GpG or GMP (100 µM). (B) Fraction bound of 32P-pGpG to 200 nM Orn in presence of no competitor, 100 µM pGpG or 100 µM GpG. (C) Fraction bound of 32P-pAp to different concentration of OrnVc was determined by DRaCALA. Kd could not be measured as 32P-pAp binding to Orn was not saturated. (B) (C) All data shown represent duplicate-independent experiments.

https://doi.org/10.7554/eLife.46313.018
Figure 5 with 1 supplement
Orn acts as a diribonucleotidase in cell lysates.

Degradation of 32P-GG (A) and 32P-AAAAAGG (B) by whole cell lysates of wild-type, orn mutant, orn mutant complemented with ornVc, or ornVc D12A. For ∆orn and ∆orn complemented with ornVc D12A,100 nM of purified OrnVc was added at 30 min time point and incubated for an additional 10 min. Samples were stopped at the indicated time and analyzed by 20% denaturing PAGE. Representative gel images of triplicated assays are shown with the indicated RNA size. Graphs show quantitation of triplicate data for indicated RNA species over time.

https://doi.org/10.7554/eLife.46313.021
Figure 5—figure supplement 1
Catalytic residues of OrnVc are required for degradation for 32P-AAAAAGG in whole-cell lysates.

Degradation of 32P-AAAAAGG by whole-cell lysates of the indicated strain (PA14, ∆orn mutant complemented with pMMB-ornVc L18A or pMMB-ornVc H158A. For ∆orn expressing ornVc L18A or ornVc H158A, 100 nM of purified, wild-type OrnVc protein was added at the 30 min time point and reactions were stopped 10 min thereafter. Samples were stopped at indicated time and analyzed by denaturing 20% PAGE. Representative gel images of two independent experiments are shown.

https://doi.org/10.7554/eLife.46313.022
Figure 6 with 2 supplements
Small-colony phenotype of ∆orn is independent of c-di-GMP signaling.

Bacterial cultures were diluted and dripped on LB agar plates with the indicated concentration of IPTG. After overnight incubation, representative images of the plates were taken and shown for (A) PA14 and PA14 ∆pelA harboring pMMB vector with the indicated diguanylate cyclase gene and (B) PA14 ∆orn and PA14 ∆ornpelA-G with pMMB and pMMB-orn. Experiments were performed in triplicate.

https://doi.org/10.7554/eLife.46313.024
Figure 6—figure supplement 1
Small-colony phenotype of ∆orn is complemented by functional ornVc alleles.

Bacterial cultures were diluted, 10 µL was spotted on LB agar plates with the indicated concentration of IPTG and drip to form parallel lines of bacteria. After overnight incubation, representative images of the plates were taken of PA14 ∆orn and PA14 ∆ornpelA-G with pMMB alone or containing the indicated allele of ornVc. Experiments were performed in triplicate and one representative image of the plates is shown.

https://doi.org/10.7554/eLife.46313.025
Figure 6—figure supplement 2
P. aeruginosa PAK ∆orn mutants show a small-colony phenotype.

Indicated bacterial strains were diluted and dripped on (A) LB agar plates or (B) LB agar plates containing 50 µg/mL carbenicillin with indicated concentration of IPTG and incubated overnight at 37°C. All experiments were performed in triplicate and one representative image of the plates is shown.

https://doi.org/10.7554/eLife.46313.026
Model for Orn’s cellular function as a diribonucleotidase.

RNA degradation is initiated by fragmentation via endoribonucleases (e.g. RNase E and RNase III) that cleave unstructured or structured RNA sequences. RNA fragments are processed further at their 3’ termini by 3’−5’ exoribonucleases (e.g. PNPase, RNase R, and RNase II). Their combined activity produces mononucleotides and various terminal diribonucleotides from the original RNA fragments. The pGpG (GG) linear diribonucleotide is also produced by linearization of c-di-GMP by specific phosphodiesterases, EAL- and HD-GYP-domain-containing enzymes, which terminate c-di-GMP signaling. In Pseudomonas aeruginosa and likely other organisms that rely on Orn for growth, Orn is the only diribonucleotidase that cleaves diribonucleotides to mononucleotides. In the absence of Orn, diribonucleotides accumulate with a drastic impact on cellular physiology, ranging from transcriptional changes, small-colony phenotypes and growth arrest, depending on the organism. Orn is also unique because it acts as the second phosphodiesterase in the degradation of c-di-GMP by clearing the pGpG intermediate. In an orn mutant, c-di-GMP levels are elevated through feedback inhibition of the c-di-GMP-degrading phosphodiesterases by pGpG, leading to the associated biofilm phenotypes.

https://doi.org/10.7554/eLife.46313.027
Author response image 1
Additives to cell lystaes do not contribute to cleavage of 32P-AAAAAGG oligoribonucleotide.

10 µg/ml DNase, 25 µg/ml lysozyme, 1 mM PMSF, and the combination of all three additives were test for cleavage of 32P-AAAAAGG. The samples were stopped at indicated times and analyzed by 20% Urea gel. All data shown represent duplicate independent experiments.

https://doi.org/10.7554/eLife.46313.031

Tables

Table 1
Quantitative measurement of length-dependent oligoribonucleotide affinities.
https://doi.org/10.7554/eLife.46313.002
SubstrateaDissociation constant Kd (nM)b
GG90 -/+9
AGG630 -/+80
AAGG890 -/+90
AAAGG2,560 -/+170
AAAAGG3,830 -/+370
AAAAAGG3,750 -/+530
  1. a: RNAs were labeled with 32P at their 5’ end. b: Affinities were measured using an established binding assay for radiolabeled RNAs (Roelofs et al., 2011; Patel et al., 2014).

Key resources table
Reagent type
or resource
DesignationSource or
reference
IdentifiersAdditional
information
Strain (P. aeruginosa)PA14Rahme et al., 1995; PMID 7604262
Strain (P. aeruginosa)PA14 ∆pelALee et al., 2007; PMID: 17824927
Strain (P. aeruginosa)PA14 ∆ornOrr et al., 2015; PMID: 26305945
Strain (P. aeruginosa)PA14 ∆orn ∆pelA-GOrr et al., 2015; PMID: 26305945
Strain (P. aeruginosa)PAKBradley, 1974; PMID: 4206974
Strain (P. aeruginosa)PAK ∆ornThis studyGenerated using pEX-Gn-∆orn (PAK)
Strain (E. coli)XL10-GoldAgilent
Strain (E. coli)Stellar cells Takara/Clontech
Strain (E. coli)BL21(DE3)New England Biolabs
Strain (E. coli)NEB T7IqNew England Biolabs
Genetic reagent (plasmid)pET28-His6-SUMO-OrnVcThis studycloned from custom DNA fragment (see below) for purification of His6-SUMO-OrnVc, OrnVc
Genetic reagent (plasmid)pET28-His6-SUMO-Rexo2This studycloned from custom DNA fragment (see below) for purification of His6-SUMO-Rexo2, Rexo2
Genetic reagent (plasmid)pDONR221-VC0341 (ornVc)Rolfs et al., 2008; PMID: 18337508
Genetic reagent (plasmid)pDONR221-ornVc D12AThis studysite-directed mutagenesis to introduce alanine at D12 position
Genetic reagent (plasmid)pDONR221-ornVc L18AThis studysite-directed mutagenesis to introduce alanine at L18 position
Genetic reagent (plasmid)pDONR221-ornVc D59AThis studysite-directed mutagenesis to introduce alanine at D59 position
Genetic reagent (plasmid)pDONR221-ornVc Q111AThis studysite-directed mutagenesis to introduce alanine at Q111 position
Genetic reagent (plasmid)pDONR221-ornVc R130AThis studysite-directed mutagenesis to introduce alanine at R130 position
Genetic reagent (plasmid)pDONR221-ornVc Y129AThis studysite-directed mutagenesis to introduce alanine at Y129 position
Genetic reagent (plasmid)pDONR221-ornVc Y129WThis studysite-directed mutagenesis to introduce tryptophan at Y129 position
Genetic reagent (plasmid)pDONR221-ornVc W61AThis studysite-directed mutagenesis to introduce alanine at W61 position
Genetic reagent (plasmid)pDONR221-ornVc W61YThis studysite-directed mutagenesis to introduce tyrosine at W61 position
Genetic reagent (plasmid)pDONR221-ornVc H66AThis studysite-directed mutagenesis to introduce alanine at H66 position
Genetic reagent (plasmid)pDONR221-ornVc H158AThis studysite-directed mutagenesis to introduce alanine at H158 position
Genetic reagent (plasmid)pVL847-ornVcOrr et al., 2015; PMID: 26305945OrnVc from pDONR cloned into pVL847 for purification of His-MBP-OrnVc, OnrVc
Genetic reagent (plasmid)pVL847-ornVc D12AThis studyD12A from pDONR cloned into pVL847 for purification of His-MBP-OrnVc D12A, OrnVc D12A
Genetic reagent (plasmid)pVL847-ornVc L18AThis studyL18A from pDONR cloned into pVL847 for purification of His-MBP-OrnVc L18A, OrnVc L18A
Genetic reagent (plasmid)pVL847-ornVc D59AThis studyL18A from pDONR cloned into pVL847 for purification of His-MBP-OrnVc D59A, OrnVc D59A
Genetic reagent (plasmid)pVL847-ornVc Q111AThis studyQ111A from pDONR cloned into pVL847 for purification of His-MBP-OrnVc Q111A, OrnVc Q111A
Genetic reagent (plasmid)pVL847-ornVc R130AThis studyR130A from pDONR cloned into pVL847 for purification of His-MBP-OrnVc R130A, OrnVc R130A
Genetic reagent (plasmid)pVL847-ornVc Y129AThis studyY129A from pDONR cloned into pVL847 for purification of His-MBP-OrnVc Y129A, OrnVc Y129A
Genetic reagent (plasmid)pVL847-ornVc W61AThis studyW61A from pDONR cloned into pVL847 for purification of His-MBP-OrnVc W61A, OrnVc W61A
Genetic reagent (plasmid)pVL847-ornVc H66AThis studyH66A from pDONR cloned into pVL847 for purification of His-MBP-OrnVc H66A, OrnVc H66A
Genetic reagent (plasmid)pVL847-ornVc H158AThis studyH158A from pDONR cloned into pVL847 for purification of His-MBP-OrnVc H158A, OrnVc H158A
Genetic reagent (plasmid)pMMB-GnFürste et al., 1986; PMID: 3549457
Genetic reagent (plasmid)pMMB-Gn-PA1107Kulasakara et al., 2006; PMID: 16477007
Genetic reagent (plasmid)pMMB-Gn-PA1120Kulasakara et al., 2006; PMID: 16477007
Genetic reagent (plasmid)pMMB-Gn-PA3702 (wspR)Kulasakara et al., 2006; PMID: 16477007
Genetic reagent (plasmid)pMMB-ApFürste et al., 1986; PMID: 3549457
Genetic reagent (plasmid)pMMB-Ap-VC0341 (ornVc)This studyOrn from pDONR cloned into pMMB for expressing in P. aeruginosa
Genetic reagent (plasmid)pMMB-Ap-ornVc D12AThis studyD12A from pDONR cloned into pMMB for expressing in P. aeruginosa
Genetic reagent (plasmid)pMMB-Ap-ornVc L18AThis studyL18A from pDONR cloned into pMMB for expressing in P. aeruginosa
Genetic reagent (plasmid)pMMB-Ap-ornVc Q111AThis studyQ111A from pDONR cloned into pMMB for expressing in P. aeruginosa
Genetic reagent (plasmid)pMMB-Ap-ornVc R130AThis studyR130A from pDONR cloned into pMMB for expressing in P. aeruginosa
Genetic reagent (plasmid)pMMB-Ap-ornVc Y129AThis studyY129A from pDONR cloned into pMMB for expressing in P. aeruginosa
Genetic reagent (plasmid)pMMB-Ap-ornVc Y129WThis studyY129W from pDONR cloned into pMMB for expressing in P. aeruginosa
Genetic reagent (plasmid)pMMB-Ap-ornVc W61AThis studyW61A from pDONR cloned into pMMB for expressing in P. aeruginosa
Genetic reagent (plasmid)pMMB-Ap-ornVc W61YThis studyW61Y from pDONR cloned into pMMB for expressing in P. aeruginosa
Genetic reagent (plasmid)pMMB-Ap-ornVc H66AThis studyH66A from pDONR cloned into pMMB for expressing in P. aeruginosa
Genetic reagent (plasmid)pMMB-Ap-ornVc H158AThis studyH158A from pDONR cloned into pMMB for expressing in P. aeruginosa
Genetic reagent (plasmid)pEX-Gn-∆orn (PAK)This study
Genetic reagent (plasmid)pJHA-GnThis study
Genetic reagent (plasmid)pJHA-Gn-ornPaThis studyOrnPA (Orr, et al, PNAS 2015) cloned into pJHA for expressing in P. aeruginosa
Genetic reagent (plasmid)pJHA-Gn-ornVcThis studyOrn from pDONR cloned into pJHA for expressing in P. aeruginosa
Genetic reagent (plasmid)pJHA-Gn-ornVc D12AThis studyD12A from pDONR cloned into pJHA for expressing in P. aeruginosa
Genetic reagent (plasmid)pJHA-Gn-ornVc L18AThis studyL18A from pDONR cloned into pJHA for expressing in P. aeruginosa
Genetic reagent (plasmid)pJHA-Gn-ornVc D59AThis studyQ111A from pDONR cloned into pJHA for expressing in P. aeruginosa
Genetic reagent (plasmid)pJHA-Gn-ornVc W61AThis studyR130A from pDONR cloned into pJHA for expressing in P. aeruginosa
Genetic reagent (plasmid)pJHA-Gn-ornVc H66AThis studyY129A from pDONR cloned into pJHA for expressing in P. aeruginosa
Genetic reagent (plasmid)pJHA-Gn-ornVc Q111AThis studyW61A from pDONR cloned into pJHA for expressing in P. aeruginosa
Genetic reagent (plasmid)pJHA-Gn-ornVc R130AThis studyH66A from pDONR cloned into pJHA for expressing in P. aeruginosa
Genetic reagent (plasmid)pJHA-Gn-ornVc H158AThis studyH158A from pDONR cloned into pJHA for expressing in P. aeruginosa
AntibodyMouse anti-HA monoclonal antibodySigma; catalog number H96581:5000
AntibodyGoat Anti-Mouse IgG-HRP conjugateSigma; catalog number A99171:5000
DNA fragment5'-GGATCCATGGCAGCCGGTGAAAG
CATGGCACAGCGTATGGTTTGGGTT
GATCTGGAAATGACCGGTCTGGATA
TTGAAAAAGATCAGATTATTGAAATG
GCCTGCCTGATTACCGATAGCGATCT
GAATATTCTGGCAGAAGGTCCGAAT
CTGATTATCAAACAGCCGGATGAACT
GCTGGATAGCATGAGCGATTGGTGT
AAAGAACATCATGGTAAAAGCGGTCT
GACCAAAGCAGTTAAAGAAAGCACCA
TTACACTGCAGCAGGCCGAATATGAAT
TTCTGAGCTTTGTTCGTCAGCAGACC
CCTCCGGGTCTGTGTCCGCTGGCAGG
TAATAGCGTTCATGAAGATAAAAAGTT
TCTGGATAAGTATATGCCGCAGTTTAT
GAAGCATCTGCATTATCGCATTATTGAT
GTGAGCACCGTTAAAGAACTGTGTCGT
CGTTGGTATCCGGAAGAATATGAGTTT
GCACCGAAAAAAGCAGCAAGCCATCGT
GCACTGGATGATATTAGCGAAAGCATC
AAAGAGCTGCAGTTTTATCGCAACAAC
ATCTTCAAAAAGAAAATCGACGAGAAAA
AACGCAAAATCATCGAAAACGGCGAAAA
CGAAAAAACCGTTAGCTAAGCGGCCGC-3'
GeneArtcustom DNA fragment for REXO2 that is codon-optimized for E. coli
DNA fragment5'-GGATCCATGAGCTTTAGCGATCAGAAT
CTGATTTGGATTGATCTGGAAATGACCG
GTCTGGACCCGGAAATGCATAAAATCAT
TGAAATGGCAACCATCGTGACCGATAGCG
AACTGAATATTCTGGCAGAAGGTCCGGTT
ATTGCAATTCATCAGCCGGAAAGCGAACT
GGCAAAAATGGATGAATGGTGTACCACCA
CCCATACCGCAAGCGGTCTGGTTGCACGT
GTTCGTCAGAGCCAGGTTAGCGAAGAAGA
AGCAATTGATCAGACCCTGGCATTTCTGAA
ACAGTGGGTTCCGGAAGGTAAAAGCCCGAT
TTGTGGTAATAGCATTGGTCAGGATCGTCG
CTTTCTGTATAAACATATGCCTCGTCTGGAA
GCCTATTTCCATTATCGTTATATTGATGTGAG
CACCATCAAAGAACTGACCCGTCGTTGGCAG
CCGGAAGTTCTGAAAGAATTTAGCAAAACCG
GTAGCCATCTGGCACTGGATGATATTCGTGA
AAGCATTGCAGAGCTGCAGTTTTATCGTAAA
GCCGTGTTTAAAATCTAAGCGGCCGC-3'
GeneArtcustom DNA fragment for OrnVc that is codon-optimized for E. coli
RNA primer5'-GG-3'Sigma
RNA primer5'-AGG-3'Sigma
RNA primer5'-AAGG-3'Sigma
RNA primer5'-AAAGG-3'Sigma
RNA primer5'-AAAAGG-3'Sigma
RNA primer5'-AAAAAGG-3'Sigma
RNA primer5'-pGG-3'Biolog; catalog number P023-01
RNA primer5'-pAA-3'Biolog; catalog number P033-01
RNA primer5'-pAG-3'GE Healthcare Dharmacon
RNA primer5'-pGA-3'GE Healthcare Dharmacon
RNA primer5'-pGC-3'GE Healthcare Dharmacon
RNA primer5'-pCG-3'GE Healthcare Dharmacon
RNA primer5'-pCU-3'GE Healthcare Dharmacon
SoftwarePrismGraphPad
SoftwareXDSKabsch, 2010; PMID: 20124693Distributed through SBGrid
SoftwarePointlessEvans, 2006; PMID: 16369096Distributed through SBGrid
SoftwareScalaEvans, 2006; PMID: 16369096Distributed through SBGrid
SoftwarePhenixAdams et al., 2010; PMID: 20124702Distributed through SBGrid
SoftwareCootEmsley et al., 2010; PMID: 20383002Distributed through SBGrid
SoftwarePymolSchrödinger
SoftwareFujifilm Multi Gauge software v3.0Fujifilm

Data availability

The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.rcsb.org (PDB ID codes 6N6A, 6N6C, 6N6D, 6N6E, 6N6F, 6N6G, 6N6H, 6N6I, 6N6J, and 6N6K). Source data files have been provided for Figures.

The following data sets were generated
  1. 1
    Protein Data Bank
    1. JD Lormand
    2. H Sondermann
    (2019)
    ID 6N6A. Vibrio cholerae Oligoribonuclease bound to pGG.
  2. 2
    Protein Data Bank
    1. JD Lormand
    2. H Sondermann
    (2019)
    ID 6N6C. Vibrio cholerae Oligoribonuclease bound to pAA.
  3. 3
    Protein Data Bank
    1. JD Lormand
    2. H Sondermann
    (2019)
    ID 6N6D. Vibrio cholerae Oligoribonuclease bound to pAG.
  4. 4
    Protein Data Bank
    1. JD Lormand
    2. H Sondermann
    (2019)
    ID 6N6E. Vibrio cholerae Oligoribonuclease bound to pGA.
  5. 5
    Protein Data Bank
    1. JD Lormand
    2. H Sondermann
    (2019)
    ID 6N6F. Vibrio cholerae Oligoribonuclease bound to pGC.
  6. 6
    Protein Data Bank
    1. JD Lormand
    2. H Sondermann
    (2019)
    ID 6N6G. Vibrio cholerae Oligoribonuclease bound to pCG.
  7. 7
    Protein Data Bank
    1. JD Lormand
    2. H Sondermann
    (2019)
    ID 6N6H. Vibrio cholerae Oligoribonuclease bound to pCpU.
  8. 8
    Protein Data Bank
    1. JD Lormand
    2. H Sondermann
    (2019)
    ID 6N6I. Human REXO2 bound to pGG.
  9. 9
    Protein Data Bank
    1. JD Lormand
    2. H Sondermann
    (2019)
    ID 6N6J. Human REXO2 bound to pAA.
  10. 10
    Protein Data Bank
    1. JD Lormand
    2. H Sondermann
    (2019)
    ID 6N6K. Human REXO2 bound to pAG.

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