1. Biochemistry and Chemical Biology
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Crystallographic observation of nonenzymatic RNA primer extension

  1. Wen Zhang
  2. Travis Walton
  3. Li Li
  4. Jack W Szostak  Is a corresponding author
  1. Howard Hughes Medical Institute, Massachusetts General Hospital, United States
  2. Harvard Medical School, United States
  3. Massachusetts General Hospital, United States
Research Article
Cite this article as: eLife 2018;7:e36422 doi: 10.7554/eLife.36422
4 figures, 2 tables, 10 data sets and 2 additional files

Figures

Chemical structures of mononucleotide and dinucleotide molecules in this study.

(A) guanosine-5′-phosphoro-2-aminoimidazolide (2-AIpG). (B) 2-aminoimidazolium-bridged guanosine dinucleotide intermediate (Gp-AI-pG). (C) 2'-deoxyguanosine-5'-monophosphate (dGMP). (D) Schematic of the RNA-dGMP complex used for crystallographic studies. The four italic nucleotides at the 5′-end represent locked nucleic acid.

https://doi.org/10.7554/eLife.36422.003
Figure 2 with 1 supplement
Crystal structure of RNA-dGMP complex.

(A) One dimeric RNA duplex (purple with yellow backbone) is bound by two dGMP monomers (cyan) at each end. (B) dGMP monomers in the +1 and+2 position of the template form Watson-Crick base pairs. Wheat meshes indicate the corresponding Fo-Fc omit maps contoured at 2.0 σ. (C) A Mg2+ ion with coordinating water molecules form extended contacts between the 3′ end of the primer and both template-bound monomers through hydrogen bonds with N7. Wheat meshes indicate the corresponding Fo-Fc omit maps contoured at 1.0 σ. (D) Top and side views of RNA-dGMP complex packing in crystal. Molecular packing of the RNA-dGMP complex shows a possible channel (dashed circle and oval) for diffusion of small molecules through the crystal.

https://doi.org/10.7554/eLife.36422.004
Figure 2—figure supplement 1
A Sr2+ ion and coordinating water molecule form an extended interaction network between the 3′ end of the primer and monomers in the +1 and+2 positions.

(A) Similar to the RNA-dGMP complex, the RNA duplex (red with yellow backbone) crystallizes with two bound GMP monomers (cyan) at each end. (B) Close up view of the template-bound GMP monomers showing a Sr2+ ion (blue sphere) with a coordinating water molecule (red sphere) interacting with the N7 of monomers in the +1 and+2 position. Wheat mesh indicates the corresponding Fo-Fc omit map contoured at 4.0 σ. Interaction distances are labeled.

https://doi.org/10.7554/eLife.36422.005
Figure 3 with 1 supplement
Template-bound Gp-AI-pG is pre-organized for phosphodiester bond formation.

(A) Structure of RNA duplex (purple with yellow backbone) bound by two Gp-AI-pG molecules (cyan) obtained after 4 hr of soaking the RNA-dGMP complex in buffer containing Gp-AI-pG. (B) Close-up of template-bound Gp-AI-pG modeled with the 2-amino group of the imidazolium bridge pointing toward the major groove and (C) minor groove. Distances for hydrogen bonds and in-line attack by the primer 3′-hydroxyl are indicated by dashed lines. Wheat meshes indicate the corresponding Fo-Fc omit maps contoured at 1.5 σ.

https://doi.org/10.7554/eLife.36422.006
Figure 3—figure supplement 1
Decay of the Gp-AI-pG intermediate in crystal soaking buffer.

31P NMR spectra of 3.5 mM Gp-AI-pG after 10 min (A) or 4 hr (B) of incubation in crystal soaking buffer (5% v/v (+/-)−2-methyl-2, 4-pentanediol, 20 mM sodium cacodylate pH 7.0, 6 mM spermine tetrahydrocholoride, 40 mM sodium chloride). The peak at −12.91 ppm corresponds to Gp-AI-pG, and −12.26 ppm to 2-AIpG. The peak at 6.01 ppm may correspond to reaction with the buffer. The peak for GMP is too broad to be observed at these concentrations. (C) Quantification of the decay of Gp-AI-pG in a pseudo-first order rate plot. The slope is equal to the negative of the first order rate constant k = (6.8 ± 0.1) x 10−2 h−1. This corresponds to a half-life of 10.2 ± 0.2 hr under these conditions. The experiment was performed in triplicate.

https://doi.org/10.7554/eLife.36422.007
Figure 4 with 1 supplement
Observation of imidazolium-bridge and phosphodiester bond formation within crystals soaked with 2-AIpG.

Wheat meshes in all structures indicate the corresponding Fo-Fc omit maps contoured at 1.5 σ. (A) Structure after 5 min of soaking shows 2-AIpG monomer binding template through Watson-Crick and non-canonical base pairing. Inset also shows electron density belonging to 2′-hydroxyl (red dashed circle) and water molecules (red dots) that interact with N7 guanosine bases. (B) Structure after 15 min soaking shows both 2-AIpG monomers bound through Watson-Crick base pairing. Inset shows two water molecules that bridge the first monomer to the primer. (C) Structures after 30 min and (D) 1 hr of soaking shows the imidazolium-bridged Gp-AI-pG intermediate bound to the template. (E) Structures after 1.5 hr, and (F) 2 hr show a mix of template-bound Gp-AI-pG and extended primer (red dashed circle) (G) After 3 hr soaking, the electron density predominantly corresponds to the extension product.

https://doi.org/10.7554/eLife.36422.009
Figure 4—figure supplement 1
Decay of the 2-AIpG monomer in crystal soaking buffer.

31P NMR spectra of 20 mM 2-AIpG after 3 min (A) or 3 hr (B) of incubation in crystal soaking buffer (5% v/v (+/-) −2-methyl-2, 4-pentanediol, 20 mM sodium cacodylate pH 7.0, 6 mM spermine tetrahydrocholoride, 40 mM sodium chloride) with 20 mM MgCl2. The large peak at −11.11 ppm corresponds to 2-AIpG. During the three hours of incubation, only 7.8% of the monomer decays, mostly to an unknown product corresponding to the peak at 5.99 ppm that may represent reaction with a buffer component. The small peak at −12.92 ppm corresponds to Gp-AI-pG. The experiment was performed in triplicate.

https://doi.org/10.7554/eLife.36422.010

Tables

Table 1
Crystallographic and structural features of RNA-ligand complexes.
https://doi.org/10.7554/eLife.36422.008
RNA- dGMPRNA- Gp-AI-pGTime-resolved structures (RNA-2-AIpG)
Stage 1 (5 min)Stage 2 (15 min)Stage 3 (30 min)Stage 4 (1 h)Stage 5 (1.5 h)Stage 6 (2 h)Stage 7 (3 h)
PDB code6C8D6C8E6C8I6C8J6C8K6C8L6C8M6C8N6C8O
Ligand binding modesWatson-CrickWatson-CrickmixedWatson-CrickWatson-CrickWatson-CrickWatson-CrickWatson-CrickWatson-Crick
N7 interactionMg2+N.D.H2OH2ON.D.N.D.N.D.N.D.N.D.
3ʹ-O-P distance, Å~6.14.63.7–4.1~5.0~4.34.1–5.01.6–4.21.6–4.11.6
P-P distance between monomers, ÅN.D.~5.1N.D.6.25.25.2–5.45.5–7.15.0–7.3~6.2
N7-N7 distance between primer and + 1 monomer, Å4.23.7–4.03.9–4.14.03.7–4.03.7–4.23.7–4.33.7–4.14.1
  1. N.D.: not detectable

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional
information
Sequence-based reagentsynthetic RNAExiqon Inc.
Commercial kitCrystallization Screening kitsHampton research, Inc.
SoftwareRefmac5University of Cambridge
DOI: 10.1107/S0907444996012255
RRID:SCR_014225
SoftwareHKL2000HKL Research Inc. DOI: 10.1016/S0076-6879(97)76066-XRRID:SCR_015547
SoftwarePhaser 2.7University of Cambridge DOI: 10.1107/S0021889807021206RRID:SCR_014219
SoftwareMestReNovaMestrelab Research, Inc.
SoftwarePymol2Schrödinger, Inc.RRID:SCR_000305

Data availability

Diffraction data have been deposited in PDB under the accession code 6C8D, 6C8E, 6C8I, 6C8J, 6C8K, 6C8L, 6C8M, 6C8N, 6C8O, 6CAB.

The following data sets were generated
  1. 1
    RNA-dGMP complex with Mg ion
    1. Zhang W
    2. Szostak JW
    (2018)
    Publicly available at the RCSB Protein Data Bank (accession no: 6C8D).
  2. 2
    RNA-imidazolium-bridged intermediate complex, 4h soaking
    1. Zhang W
    2. Szostak JW
    (2018)
    Publicly available at the RCSB Protein Data Bank (accession no: 6C8E).
  3. 3
    RNA-activated 2-AIpG monomer complex, 5 min soaking
    1. Zhang W
    2. Szostak JW
    (2018)
    Publicly available at the RCSB Protein Data Bank (accession no: 6C8I).
  4. 4
    RNA-activated 2-AIpG monomer complex, 15 min soaking
    1. Zhang W
    2. Szostak JW
    (2018)
    Publicly available at the RCSB Protein Data Bank (accession no: 6C8J).
  5. 5
    RNA-activated 2-AIpG monomer complex, 30 min soaking
    1. Zhang W
    2. Szostak JW
    (2018)
    Publicly available at the RCSB Protein Data Bank (accession no: 6C8K).
  6. 6
    RNA-activated 2-AIpG monomer complex, 1h soaking
    1. Zhang W
    2. Szostak JW
    (2018)
    Publicly available at the RCSB Protein Data Bank (accession no: 6C8L).
  7. 7
    RNA-activated 2-AIpG monomer, 1.5h soaking
    1. Zhang W
    2. Szostak JW
    (2018)
    Publicly available at the RCSB Protein Data Bank (accession no: 6C8M).
  8. 8
    RNA-activated 2-AIpG monomer complex, 2h soaking
    1. Zhang W
    2. Szostak JW
    (2018)
    Publicly available at the RCSB Protein Data Bank (accession no: 6C8N).
  9. 9
    RNA-activated 2-AIpG monomer, 3h soaking
    1. Zhang W
    2. Szostak JW
    (2018)
    Publicly available at the RCSB Protein Data Bank (accession no: 6C8O).
  10. 10
    RNA-dGMP complex with Sr2+ ion
    1. Zhang W
    2. Szostak JW
    (2018)
    Publicly available at the RCSB Protein Data Bank (accession no: 6CAB).

Additional files

Supplementary file 1

Table S1: Data collection statistics.

Table S2: Structure refinement statistics.

https://doi.org/10.7554/eLife.36422.011
Transparent reporting form
https://doi.org/10.7554/eLife.36422.012

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