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Molecular basis for noncanonical transcription initiation from Np4A alarmones

Nature Chemical Biology (2025)Cite this article

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Abstract

Stress-induced dinucleoside tetraphosphates (Np4Ns, where N is adenosine, guanosine, cytosine or uridine) are ubiquitous in living organisms, yet their function has been largely elusive for over 50 years. Recent studies have revealed that RNA polymerase can influence the cellular lifetime of transcripts by incorporating these alarmones into RNA as 5′-terminal caps. Here we present structural and biochemical data that reveal the molecular basis of noncanonical transcription initiation from Np4As by Escherichia coli and Thermus thermophilus RNA polymerases. Our results show the influence of the first two nucleotide incorporation steps on capping efficiency and the different interactions of Np4As with transcription initiation complexes. These data provide critical insights into the substrate selectivity that dictates levels of Np4 capping in bacterial cells.

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Fig. 1: Np4Ns used in this study and their comparative efficiency of incorporation.
Fig. 2: Structures of T. thermophilus transcription initiation complexes.
Fig. 3: Structures of T. thermophilus transcription initiation complexes containing Np-capped dinucleotide RNAs.
Fig. 4: Structure of the T. thermophilus transcription initiation complex containing Gp4-capped trinucleotide RNAs and mutations in E. coli RNAP to assess capping efficiency.
Fig. 5: MD simulations of T. thermophilus transcription initiation complexes.
Fig. 6: Schematic summarizing the incorporation of Np4As during transcription initiation from promoters with different nucleotides at the −1 position of the template DNA strand.

Data availability

Coordinates of the structures and the cryo-EM density maps were deposited to the PDB and the EM Data Bank under the following accession codes: 9UKN and EMD-64250, Apo(−1dC); 9UJK and EMD-64215, ATP(−1dC); 9UJL and EMD-64216, Ap4A(−1dC); 9UPW and EMD-64404, Gp4A(−1dC); 9UKS and EMD-64253, Gp4A(−1dA); 9UJP and EMD-64219, Up4A(−1dA); 9UJN and EMD-64218, ATP-C(−1dC); 9UKP and EMD-64252; Ap4A-C(−1dC); 9UKU and EMD-64255, Gp4A-C(−1dC); 9UKO and EMD-64251, Ap4A-C(−1dA); 9UMA and EMD-64271, Gp4A-C(−1dA); 9ULS and EMD-64266, Up4A-C*(−1dA); 9UKT and EMD-64254, Gp4A-C-U(−1dC); 9ULT and EMD-64267, Up4A-C(−1dA); 9V4M, Ap4G(−1dG). The MD simulation data used in this study are available from Zenodo (https://doi.org/10.5281/zenodo.15493272)72. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Institutes of Health (NIH) grants T32 GM088118-09 to J.W.W., R35GM145359 and R01GM035769 to J.G.B., R01GM147652 to X.H. and R01GM112940 and R21GM151508 to A.S. The work was also supported by the Blavatnik Family Foundation and by the Howard Hughes Medical Institute (E.N.), the Hirschfelder Professorship Fund and the Research Forward Fund from the University of Wisconsin-Madison (X.H.) and the Croucher Fellowship for Postdoctoral Researchers from Croucher Foundation (I.C.U.). Some of the work was performed at NYU Langone Health’s Cryo-EM Laboratory (RRID: SCR_019202), which is partially supported by the Laura and Isaac Perlmutter Cancer Center Support Grant from the NIH National Cancer Institute (P30CA016087). The Laboratory for BioMolecular Structure is supported by the Department of Energy (DOE) Office of Biological and Environmental Research (KP1607011). The National Center for Cryo-EM Access and Training and the Simons EM Center located at the New York Structural Biology Center are supported by the NIH Common Fund Transformative High-Resolution Cryo-EM program (U24 GM129539) and by grants from the Simons Foundation (SF349247) and NY State Assembly Majority. This research used the Northeastern Collaborative Access Team beamlines, funded by the NIH (P30 GM124165), at the Advanced Photon Source, a US DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. This study used beamline 17-ID-2 (FMX) of the National Synchrotron Light Source II, a US DOE Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under contract no. DE-SC0012704. The beamline operation is supported by the Center for BioMolecular Structure, funded by the NIH (P30GM133893) and the DOE Office of Biological and Environmental Research (FWP BO070). We thank L. Li for helping to purify Tt-RNAP and V. Epstein for discussions.

Author information

Author notes

  1. These authors contributed equally: Wenqian Duan, Abhishek Kaushik.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY, USA

    Wenqian Duan, Abhishek Kaushik, Mengjie M. J. Liu, Jacob W. Weaver, Evgeny Nudler & Alexander Serganov

  2. Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA

    Ilona C. Unarta, Yue Wu & Xuhui Huang

  3. Division of Advanced Research Technologies, New York University School of Medicine, New York, NY, USA

    Bing Wang & William J. Rice

  4. Department of Cell Biology, New York University School of Medicine, New York, NY, USA

    William J. Rice

  5. Department of Microbiology, New York University School of Medicine, New York, NY, USA

    Daniel J. Luciano & Joel G. Belasco

  6. Howard Hughes Medical Institute, New York University School of Medicine, New York, NY, USA

    Evgeny Nudler

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  1. Wenqian Duan
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Contributions

W.D. carried out the structural studies on Tt-RNAP and contributed to the biochemical experiments. A.K. performed the in vitro transcription assays and contributed to the structural studies. I.C.U., Y.W. and X.H. conducted the MD simulations. J.W.W. conducted the structural studies on Ec-RNAP. B.W., W.J.R. and M.M.J.L. participated in the cryo-EM data collection and processing. W.D., J.G.B., D.J.L., X.H., E.N. and A.S. planned and interpreted the experiments. A.S. supervised the project. W.D., A.K., J.G.B. and A.S. wrote the paper with input from all the authors.

Corresponding author

Correspondence to Alexander Serganov.

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Extended data

Extended Data Fig. 1 Efficiency of Np4A incorporation during transcription initiation.

a-c, Determination of the relative efficiencies of transcription initiation with Ap4A vs. ATP in the first transcription step. a, Representative gel showing the initial RNA products of transcription reactions performed in the presence of 0.75 mM Ap4A and 0.008-1.8 mM ATP as the initiating nucleotides and [α32P]-CTP as the extending nucleotide. The experiment was repeated 3 times with similar results, with quantification shown in panels (b) and (c). b, Graph showing the fraction of Ap4A-initiated RNA vs. the Ap4A/ATP ratio in transcription reactions conducted with tDNAs containing various -1 nucleotides. The curves are logarithmic regression fits using the three highest concentrations of ATP (0.52, 0.90, 1.80 mM) from panel (a). Each point is the mean ± SD of three independent experiments (n = 3). c, Relative efficiencies of transcription initiation with Ap4A vs. ATP [(kcat/Km, Ap4A)/(kcat/Km, ATP)] determined from panel (b). Each bar is the mean ± SD of three independent experiments (n = 3). d, Promoter sequences used to validate that DNA templates for cryo-EM studies of Tt-RNAP show expected differences in transcription efficiency from Np4A with purines or pyrimidines at the -1 position of tDNA. Nucleotides in orange differ from the promoter in Fig. 1e. e-f, Efficiency of Np4A incorporation by Tt-RNAP from the promoter described in panel (d). The efficiency was determined as in Fig. 1. e, A representative gel used for quantification. f, A bar graph showing data points from two independent experiments (n = 2).

Source data

Extended Data Fig. 2 X-ray crystal structure of the T. thermophilus transcription initiation complex loaded with Gp4A.

a, DNA template scaffold used for crystallization. b, The overall view of the structure is shown with a simulated annealing omit map contoured at 0.9 σ level (light blue mesh). Proteins and DNA are color-coded, as shown. Gp4A is shown in surface representation (magenta). c, Zoom-in view of the Gp4A and selected surrounding residues shown with a simulated annealing composite omit map contoured at 0.9 σ level. The view is similar to (b).

Extended Data Fig. 3 Cryo-EM maps and structures of the active site of T. thermophilus transcription initiation complexes containing Np4-capped di- and trinucleotide RNAs.

The figures focus on the transcription products (initiating Np4As in magenta and other nucleotides in pink) and tDNA (colored as in Fig. 2) shown with schematics of the RNA-template interactions (top), matching maps in mesh representation (middle) and zoomed-in top views of the maps and models of the cap moieties (bottom or right). a, ATP-C(-1dC) complex. b, Gp4A-C(-1dC) complex. c, Gp4A-C(-1dA) complex. d, Ap4A-C(-1dC) complex. e, Ap4A-C(-1dC) complex. f, Up4A-C(-1dA) complex. g, Gp4A-C-U(-1dC) complex. To show map quality with reduced background, the map contour level was set higher in the zoomed-in images (bottom) than in the overall views (top) by ~10 % (b, f) or ~20 % (d, e).

Extended Data Fig. 4 MD simulations of the modelled T. thermophilus transcription initiation complexes.

a-c, MD simulations of the structure-based models of Ap4A(-1dC) (a), Up4A(-1dA) (b), and Up4A-C(-1dC) (c), and the cryo-EM structure of Up4A-C(-1dCA) (d). The left panels show two-dimensional heatmap plots of the conformations represented by the +1 dT – A base-pair geometry. The middle panels show the plots for potential base pairing between the cap nucleotide and nucleotide -1 of the tDNA. A black star corresponds to the reference Watson-Crick base-pairing. A red cross depicts the initial structure at the start of the simulation. The right panels show representative structures from various parts of the heatmap indicated by colored triangles. Initial structures are in red. e, Base-pairing distance distribution in Up4A-C(-1dC) (left) and Up4A-C(-1dA) (right) systems. The X-axis corresponds to the base-pairing distance in (c) and (d), and the Y-axis shows the corresponding densities estimated via KDE. The dashed red line indicates the position of the initial model at ~9.0 Å. The percentage indicates the number of simulation frames that are close to the ideal base-pairing (Site 1), with the cutoff distance of 9.0 Å.

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Supplementary Tables 1–5 and Figs. 1–21.

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Source Data Fig. 1

Unprocessed gels.

Source Data Fig. 4

Unprocessed gels.

Source Data Extended Data Fig. 1

Unprocessed gels.

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Duan, W., Kaushik, A., Unarta, I.C. et al. Molecular basis for noncanonical transcription initiation from Np4A alarmones. Nat Chem Biol (2025). https://doi.org/10.1038/s41589-025-02044-6

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