The development of unnatural base pairs (UBPs) has greatly increased the information storage capacity of DNA, allowing for transcription of unnatural RNA by the heterologously expressed T7 RNA polymerase (RNAP) in Escherichia coli. However, little is known about how UBPs are transcribed by cellular RNA polymerases. Here, we investigated how synthetic unnatural nucleotides, NaM and TPT3, are recognized by eukaryotic RNA polymerase II (Pol II) and found that Pol II is able to selectively recognize UBPs with high fidelity when dTPT3 is in the template strand and rNaMTP acts as the nucleotide substrate. Our structural analysis and molecular dynamics simulation provide structural insights into transcriptional processing of UBPs in a stepwise manner. Intriguingly, we identified a novel 3′-RNA binding site after rNaM addition, termed the swing state. These results may pave the way for future studies in the design of transcription and translation strategies in higher organisms with expanded genetic codes.
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This work was supported by the National Institutes of Health (nos. R01 GM102362 to D.W., GM118178 to F.E.R. and GM128376 to R.J.K.). R.K. acknowledges supported from NASA Exobiology (no. NNX14AP59G). F.E.R. acknowledges support from Synthorx, a Sanofi company. X.H. acknowledges support from the Padma Harilela Endowment fund.
The authors declare no competing interests.
Peer review information Nature Chemical Biology thanks Seth Darst, Xianyang Fang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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a, Unbiased 2Fo-Fc omit electron density map of rNaMTP is contoured at 1.2 σ. b, Unbiased 2Fo-Fc omit electron density map of rNaM is contoured at 1.2 σ.
Extended Data Fig. 2 The relative stability of NTPs to maintain good activation geometry is plotted as a function of total simulation time.
The relative stability is defined as the stability of NTPs in comparison with rNaMTP to maintain good activation geometry, -ln(pNTP/prNaMTP), in energy unit (RT). pNTP and prNaMTP are the percentage of frames with good activation in NTP and rNaMTP simulations, respectively. The criteria for good activation geometry are 3.0 Å ≤ distance between O3′ - Pα ≤ 3.5 Å and 7.0 Å ≤ base pair distance (rNaMTP, ATP or GTP) ≤ 9.0 Å, 6.0 Å ≤ base pair distance (CTP, UTP) ≤ 8.0 Å. The plot shows that the simulation has converged as the order of stability among NTPs remains the same regardless of the simulation time. Importantly, rNaMTP indeed is the most stable substrate when dTPT3 is the template DNA. The data are shown as mean values ± standard deviation, which were calculated by bootstrapping of N independent production MD simulations (N=4, 8, 12, 16 for data at the time point of 200, 400, 600, 800 ns in the x-axis, respectively).
Extended Data Fig. 3 MD simulation of ATP and rTPT3TP at A site across dNaM (with both Mg2+ ion A & Mg2+ ion B).
MD simulation of ATP and rTPT3TP at A site across dNaM (with both Mg2+ ion A & Mg2+ ion b). (a and b) Left panel: two dimensional heatmap plot of the base pairing geometry. Base pair distance is the distance between center of mass of dNaM and NTPs. We observed strong localization of simulation frames in the dNaM-rTPT3TP pair, while ATP was highly dispersed both in distance and angle. Right panel: Distance of nucleophilic attack. Distribution of simulation frames sorted by the distance between Pα of incoming NTP and O3´of terminal RNA is plotted. Good activation geometry (3.0 Å ≤ distance between O3´- Pα ≤ 3.5 Å and 6.0 Å ≤ base pair distance ≤ 8.0 Å) is indicated with red dotted lines. Percentage of simulation frames with catalytically active conformation was shown as mean values ± standard deviation, which were calculated by bootstrapping of N independent production MD simulations (N=16).
Extended Data Fig. 4 UBP structure from DNA polymerase (dNaM-d5SICSTP, PDB 3SV3) shows co-planar edge-to-edge configuration.
UBP structure from DNA polymerase (dNaM-d5SICSTP, PDB 3SV3) shows co-planar edge-to-edge configuration.
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Oh, J., Shin, J., Unarta, I.C. et al. Transcriptional processing of an unnatural base pair by eukaryotic RNA polymerase II. Nat Chem Biol 17, 906–914 (2021). https://doi.org/10.1038/s41589-021-00817-3