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Structural basis for RNA polymerase III transcription repression by Maf1

Nature Structural & Molecular Biology volume 27, pages 229–232 (2020)Cite this article

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Abstract

Maf1 is a conserved inhibitor of RNA polymerase III (Pol III) that influences phenotypes ranging from metabolic efficiency to lifespan. Here, we present a 3.3-Å-resolution cryo-EM structure of yeast Maf1 bound to Pol III, establishing that Maf1 sequesters Pol III elements involved in transcription initiation and binds the mobile C34 winged helix 2 domain, sealing off the active site. The Maf1 binding site overlaps with that of TFIIIB in the preinitiation complex.

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Fig. 1: Structure of the Maf1–Pol III complex.
Fig. 2: Structure-guided mutagenesis confirms the mechanism of transcription inhibition.

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Data availability

The cryo-EM map of the Maf1–Pol III complex has been deposited to the Electron Microscopy Data Bank (EMDB) under the accession code EMD-10595. The coordinates of the corresponding model have been deposited to the Protein Data Bank (PDB) under accession code 6TUT. Source data for Fig. 2b,c are available with the paper.

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Acknowledgements

This work was supported by an ERC Advanced Grant (ERC-2013-AdG340964-POL1PIC) to C.W.M. and R.W., a National Institutes of Health Grant (GM120358) to I.M.W. and an EMBL International PhD program award to M.K.V. We thank M. Girbig for help with transcription assays and critical reading of the manuscript and F. Weis for EM support. We are grateful to T. Hoffmann and J. Pecar for maintaining the high performance computing environment for EM data processing at EMBL.

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Authors and Affiliations

  1. Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

    Matthias K. Vorländer, Florence Baudin, René Wetzel, Wim J. H. Hagen & Christoph W. Müller

  2. Collaboration for Joint PhD Degree between EMBL and Heidelberg University Faculty of Biosciences, Heidelberg University, Heidelberg, Germany

    Matthias K. Vorländer

  3. Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA

    Robyn D. Moir & Ian M. Willis

  4. Department of Systems and Computational Biology, Albert Einstein College of Medicine, Bronx, NY, USA

    Ian M. Willis

Authors
  1. Matthias K. Vorländer
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  2. Florence Baudin
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  3. Robyn D. Moir
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  4. René Wetzel
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  5. Wim J. H. Hagen
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  6. Ian M. Willis
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  7. Christoph W. Müller
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Contributions

The project was conceived by C.W.M., I.M.W. and R.D.M. and supervised by C.W.M. M.K.V. designed, carried out and analyzed experiments, data processing and model building. F.B. performed transcription assays. W.J.H.H. collected cryo-EM data. R.W. was responsible for yeast fermentation. M.K.V. and C.W.M. prepared the manuscript with input from the other authors. R.D.M. performed functional analysis of the acidic tail.

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Correspondence to Christoph W. Müller.

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The authors declare no competing interests.

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Peer review information Beth Moorefield and Inês Chen were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Cryo-EM data quality.

Representative micrograph denoised using Warp25, 2D classes, EM density colored by local resolution, and angular distribution coverage.

Extended Data Fig. 2 Particle classification strategy and model validation.

Top panel: Micrographs were divided into four batches and classified using 3D classification in RELION with a 60 Å low-pass filtered map of Pol III as a reference. The best class of each batch was retained, batches were combined and refined. Focused classification using a mask on Maf1 yielded one class with improved Maf1 occupancy. Lastly, focused classification using a mask on the stalk-heterotrimer-clamp module separated an open clamp state with poorly resolved Maf1 density from a closed-clamp state with clear separation of the Maf1 β-strands and side-chain density. Bottom panel: FSC curves showing the correlation between independent half maps (left) and the correlation between the sharpened experimental map and a simulated model map (right).

Extended Data Fig. 3 Multiple sequence alignment of Maf1.

Shown are sequences of loop 1, loop 2 and W319 from distantly related species. Functionally important sites based on mutant studies are boxed in red.

Extended Data Fig. 4 Comparison of the Pol III conformation in apo-Pol III, Maf1-Pol III and the Pol III-PIC.

Maf1-Pol III adopts a similar conformation as the Pol III-PIC (note the distance between the lobe (blue) and clamp head (red)).

Extended Data Fig. 5 Comparison of Maf1 structures from S. cerevisiae, H. sapiens and C. sinensis.

Top panel: Structures shown in ribbon representation with α-helices colored in red and β-sheets colored in yellow. Bottom panel: surface representation colored by electrostatic potential from negative (red) to positive (blue) potential.

Extended Data Fig. 6 A model of the disordered regions in Maf1.

The phospho-regulatory region of Maf1 is accessible in the Maf1-Pol III structure, whereas the acidic tail emerges in the direction of the DNA binding cleft. The downstream DNA (PDB 6F40) has been superimposed with the Maf1-Pol III structure to help visualization. Maf1 is colored from N-terminus (blue) to C-terminus (red). Putative paths of the disordered regions are indicated, with the internal, phospho-regulated region located away from the Pol III interface, whereas the acidic C-terminal tail projects towards the DNA binding cleft, where it might help to repel nucleic acids.

Extended Data Fig. 7 The C-terminus of ScMaf1 is not obligatory for Maf1 function.

a, Amino acid sequences of wild-type ScMaf1 and the various ScMaf1 C-terminal mutants are shown from the end of conserved domain C22 through the acidic terminal region (where present). Amino acid sequences from residue 2 through 326 are represented by //. ScMaf1ΔCt terminates at residue 346 while ScMaf1ΔCtSpMAF1 and ScMaf1ΔCtHsMAF1 proteins contain the terminal amino acids from S. pombe (35 residues, colored in blue) and human (45 residues, colored in pink) MAF1 proteins appended to ScMaf1ΔCt. b, The respiratory defect of the maf1Δ::natMX vector only strain, poor growth on glycerol, is rescued by wild-type ScMaf1 and the three ScMaf1 C-terminal variants. Ten-fold serial dilutions of maf1Δ:: natMX strains containing pRS314 vector, pRS314ScMaf1 or pRS314ScMaf1 C-terminal variants grown at 30 and 37 °C on media with glucose (left panels) and glycerol (right panels) as the carbon source. c, Northern analysis of Pol III transcription and repression shows that the C-terminus of ScMaf1 is not required for the Maf1 repression function in yeast. maf1Δ:: natMX strains containing pRS314ScMaf1 or pRS314ScMaf1 C-terminal variants (ScMaf1ΔCt, ScMaf1ΔCtSpMAF1 and ScMaf1ΔCtHsMAF) were treated with rapamycin or drug vehicle for 1 h. The relative level of Pol III transcription is reported by the amount of pre-tRNALeu transcript normalized to U3 snRNA, expressed relative to the untreated wild-type strain and indicated below each lane.

Supplementary information

Supplementary Information

Cryo-EM data collection and refinement statistics.

Reporting Summary

Source data

Source Data Fig. 2

Unprocessed and uncropped gels, source data for Fig 2b,c.

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Vorländer, M.K., Baudin, F., Moir, R.D. et al. Structural basis for RNA polymerase III transcription repression by Maf1. Nat Struct Mol Biol 27, 229–232 (2020). https://doi.org/10.1038/s41594-020-0383-y

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