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Structural Studies of Eukaryotic RNA Polymerase I Using Cryo-Electron Microscopy

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Part of the Methods in Molecular Biology book series (MIMB,volume 2533)

Abstract

Technical advances have pushed the resolution limit of single-particle cryo-electron microscopy (cryo-EM) throughout the past decade and made the technique accessible to a wide range of samples. Among them, multisubunit DNA-dependent RNA polymerases (Pols) are a prominent example. This review aims at briefly summarizing the architecture and structural adaptations of Pol I, highlighting the importance of cryo-electron microscopy in determining the structures of transcription complexes.

Key words

  • RNA polymerase I
  • Cryo-electron microscopy
  • Pre-rRNA transcription

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1 Cryo-Electron Microscopy : The New Standard in Transcription Research

The visualization of macromolecular complexes is essential to our understanding of their function. This is especially true for eukaryotic RNA polymerases (Pol) I, II, and III. These enzymes play a pivotal role within the central dogma of molecular biology by synthesizing the 35S ribosomal RNA precursor (Pol I), messenger and many noncoding RNAs (Pol II), and tRNAs , 5S rRNA , U6 snRNA as well as other small, structured RNAs (Pol III).

Almost 20 years ago, advances in cryo-crystallography allowed solving the structure of RNA polymerase II , first in its 10-subunit form [1], later comprising all 12 subunits [2], providing insights into the function of this molecular machine at an unprecedented level of detail. This gave rise to a number of follow-up studies, resulting in structures of an actively elongating Pol II form [3, 4] and elongation [5, 6] or initiation [7, 8] factor–bound Pol II. Thereby, the molecular mechanisms of transcription , as well as regulatory and catalytic functions of transcription factors , could be deciphered.

However, X-ray diffraction analysis relies on the availability and quality of the analyzed crystals. It is therefore not surprising that a number of transcription factors could never be successfully studied in complex with their respective polymerase by crystallography. This includes the general Pol II initiation factors TFIIF and TFIIE, which are involved in initiation complex formation and promoter DNA melting [9]. A crystal structure of Pol I was solved 10 years after Pol II in an inactive conformation [10, 11], a Pol III crystal structure is still lacking to date. Crystallization depends on high amounts of purified material. Whereas conformational heterogeneity usually is problematic for crystal formation, cryo-EM allows for visualization of different functional states of proteins in vitrified ice, therefore capturing close-to-native states. Technical improvements such as the development of direct electron detectors [10], highly stable microscopes and improved processing software [11, 12] pushed previous limitations of the technique and led to the often quoted “resolution revolution” in cryo-EM [13]. Within this book, we describe protocols for a sample preparation and single-particle cryo-EM screening workflow, adapted to RNA polymerase complexes (Chapter 6 ).

Here, we aim to briefly outline the benefits and limitations of single-particle cryo-EM for the analysis of transcription complexes at the example of RNA polymerase I in the context of functional characterization reviewed by Merkl et al. in the same issue.

2 Pol I Specific Subunits Resemble Built-in Transcription Factors

Yeast Pol I has a molecular weight of 590 kDa and consists of 14 protein subunits. A core of ten subunits includes the large subunits A190 and A135, the subcomplex AC40/AC19 (shared with Pol III), the common subunits Rpb5 , Rpb6 , Rpb8 , Rpb10 , and Rpb12 (also included in Pol II and Pol III) and the subunit A12.2. Pol I also contains the specific subunit complex A14/A43 forming the “stalk” and the specific heterodimer subcomplex A49/A34.5. In Fig. 1, we present a “hybrid model” of Pol I, constructed using the software package COOT [14]. The model combines structural information obtained from cryo-EM reconstructions of elongation and initiation complexes and the dimer crystal structure (see below). The Pol I subunit complex A49/A34.5 structurally and functionally resembles a built-in version of subunits Tfg1/2 constituting the Pol II initiation factor TFIIF [15] and is involved in open complex stabilization as well as promoter escape. Nevertheless, subcomplex A49/A34.5 may also contribute to transcript cleavage activity [16] and may play a role in elongation [17], as reviewed by Merkl et al. in this book. The Pol I subunit A12.2 displays features of the Pol II subunit Rpb9 , as well as the Pol II elongation /cleavage factor TFIIS , explaining the intrinsic ability of Pol I for transcript cleavage [18] and efficient recovery from deep backtracks [19].

Fig. 1
figure 1

Ribbon model of RNA polymerase I highlighting specific subunits and –domains. Hybrid model constructed using COOT [14] for demonstration purposes. The structure of the Pol I elongation complex (PDB 5M3F) was extended by adding (a) a model of the C-terminal domain of subunit A12.2, (b) the “connector” domain of subunit A43 from the crystal structure (both from PDB 4C2M) and (c) the linker and tandem-winged helix domains [15] from an ITC reconstruction (PDB 5 W66). The “front view” looks along the incoming (“downstream”) DNA. Subunits not visible in the front view but present in the model are AC19, Rpb10 , and Rpb12 . Cyan spheres depict coordinated zinc atoms

The constitutive association of subunits A12.2 and A49/A34.5 may be a symptom of the enzyme’s adaptation to transcribing one specific, extraordinarily long gene. A differential regulation of Pol I transcription similar to the conditional, gene-specific regulation of Pols II and III is most likely not required. Instead, a binary activity switch can be achieved by posttranslational modification of the polymerase or its initiation factors [20,21,22,23].

3 The Pol I Transcription Cycle Visualized In Vitro

Throughout transcription of a DNA template, Pols pass through three main phases, which are more or less conserved throughout various organisms. First, the Pol is recruited to the template sequence, melts the DNA duplex with or without the help of additional factors and begins the synthesis of an RNA strand (initiation ). Thereafter, the initial RNA is extended according to the DNA template strand (elongation ). Finally, Pol dissociates from its DNA template and releases its RNA product (termination ) [24].

The Pol I transcription cycle requires few basic transcription factors [25, 26]. Basal initiation in yeast can be achieved with only the factor Rrn3 and the Core Factor (CF) complex , consisting of proteins Rrn6 , Rrn7 , and Rrn11 [27, 28]. Elongation may involve regulatory factors in vivo, but can commence in vitro without them [25, 26]. Termination finally requires a Myb-domain containing protein, Nsi1 in yeast [29, 30]. Pol I can adopt a dimeric state specific to this enzyme, [31], in which both molecules are inactivated [32] and prevented from binding initiation factors Rrn3 and CF [33,34,35]. Dimerization is reversible [33] and may play a role in storage of Pol I molecules during starvation [36, 37]. Using cryo-EM in many variations, our understanding of the Pol I transcription cycle has been significantly improved in recent years. A structural description of the Pol I transcription cycle allowed a detailed structure–function analysis and the comparison to other transcription systems [38,39,40,41].

Specifically, single-particle cryo-EM analyses showed how Pol I monomers are bound by the initiation factor Rrn3 [42], which allows for recruitment of CF subunit Rrn7 [37, 43, 44] that is related to the general Pol II initiation factor TFIIB [45, 46]. The interaction with Rrn3 prevents the formation of transcriptionally inactive Pol I dimers. In baker’s yeast (Saccharomyces cerevisiae ) Pol I dimerization depends on the specialized, nonconserved “connector” domain of subunit A43, suggesting that this mode of regulation is specific to S. cerevisiae [37]. However, recent cryo-EM studies of Pol I from fission yeast (Schizosaccharomyces pombe ) demonstrated that inactive dimers can form independent of the connector domain utilizing divergent structure elements [47]. It is therefore possible that different organisms evolved different dimer interfaces, but use the same strategy of Pol I hibernation by dimerization during periods of starvation.

Monomeric Pol I bound to Rrn3 can be recruited by promoter-engaged CF . Whereas the structure of CF was determined by X-ray crystallography [48], its interaction with the Pol I–Rrn3 complex was apparently too flexible for this method to succeed. Instead, three independent studies used cryo-EM to visualize a reconstituted initially transcribing complex (ITC) that relies on an artificially stabilized, mismatched transcription bubble with a short initial product RNA [48,49,50]. Comparison of these studies shows that seemingly similar experimental cryo-EM approaches may yield different, though highly complementary results. This demonstrates the influence of sample preparation and experimental conditions on the outcome of a cryo-EM experiment and the strength of single-particle cryo-EM to elucidate dynamic processes. Specifically, some complexes exist in conformations that fail to bind Rrn3 [49], which is required essential to the initiation process and dissociates after promoter escape [42, 51, 52]. These complexes may represent later initiation intermediates. In another structure, the RNA primer is lost and the C-terminal domain of subunit A12.2 is inserted into the funnel domain of Pol I, as would be expected during RNA cleavage events [50]. A third reconstruction showed local resolution differences suggesting high flexibility in distal CF regions [48]. More recently, consolidating structures of close-to-native preinitiation complexes were reported [53, 54]. A closed and an open complex could be reconstructed from a small fraction of recorded particles but yielded important information about the roles of a flexible loop in Rrn3 in CF engagement, and the role of the linker/tWH domain of subunit A49 in template DNA melting, open bubble stabilization, and Rrn3 dissociation [53].

Following initiation , Pol I adopts an actively elongating conformation. This conformation was never successfully crystallized but was reconstructed by single-particle cryo-EM [55, 56]. It is still under debate, whether dissociation/transient association of the A49/A34.5 heterodimer and formation of a 12-subunit polymerase has a physiological function under some circumstances. Chromatin immunoprecipitation (ChIP ) data does not suggest subunit depletion along the 35S gene body in vivo [57], although a lack of density in cryo-EM reconstructions of S. cerevisiae and S. pombe Pol I ECs suggests that subdomain relocation can take place [47, 58]. Upon initiation , the central DNA-binding cleft contracts and tightly binds downstream DNA and the DNA–RNA hybrid region, thus facilitating processive elongation . Contraction upon activation is apparently common to Pol I regulation in different organisms [47] but differs from Pol II [3] and III [59]. The various Pol I EC reconstructions demonstrated the versatile nature of the specific subunits A12.2, A49, and A34.5 [47, 55, 56, 58], and the importance of a Pol I-specific arginine residue in the “bridge-helix” in stalling at DNA lesions caused by UV radiation [60]. Furthermore, the physiological relevance of a contracted Pol I state was confirmed by the 3D-reconstruction of the enzyme from ex vivo purified, actively transcribing Pol I using electron cryo-tomography [55]. Interestingly enough, the underlying preparation technique has been used to describe Pol I functionality since the 1960s [61].

Structural information on transient and highly dynamic states of the Pol I transcription cycle , such as termination , are still lacking to date.

4 Outlook

Continuing investigation of the Pol I transcription system currently aims at defining the structural basis of promoter recruitment in the context of TATA-binding protein [62] and yeast upstream activating factor (UAF) which are important to achieve full initiation levels under physiological conditions [27, 63]. The structural investigations of these complexes and their interplay with the Pol I enzyme will be the key to understand this initiation systems in comparison with Pol II [64, 65] and Pol III [66, 67]. Ultimately, such comparative structure–function analyses will enable us to understand the particular adaptation of the Pol I transcription system to pre-rRNA transcription .

Cryo-EM has now firmly established itself as the method of choice to analyze the structure of such mega-Dalton sized, dynamic, macromolecular complexes. However, the method has also proven to be prone to certain errors. Especially the lack of tools for intrinsic validation of density interpretation may be an issue for initial sequence assignment at resolutions worse than 4 Å. Continuing method development at the experimental and the computational level continues and may soon overcome issues of flexibility and density assignment. For the time being, however, the interpretation of cryo-EM reconstructions should be carefully evaluated and supported by functional data or mutational analysis. Results can also be strengthened by crystallization or small angle X-ray scattering analysis of flexible domains [68], native mass spectrometry [15], hydrogen–deuterium exchange mass-spectrometry evaluation [69], or single-molecule FRET analysis [70]. Especially distance restrains obtained from protein crosslinking coupled to mass spectrometry provides architectural information and may assist low-resolution density assignment [71, 72].

References

  1. Cramer P, Bushnell DA, Kornberg RD (2001) Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution. Science 292:1863–1876. https://doi.org/10.1126/science.1059493

    CAS  CrossRef  PubMed  Google Scholar 

  2. Armache J-K, Mitterweger S, Meinhart A, Cramer P (2004) Structures of complete RNA polymerase II and its subcomplex, Rpb4/7. J Biol Chem 280(8):7131–7134. https://doi.org/10.1074/jbc.M413038200

  3. Gnatt AL, Cramer P, Fu J, Bushnell DA, Kornberg RD (2001) Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 A resolution. Science 292:1876–1882. https://doi.org/10.1126/science.1059495

    CAS  CrossRef  PubMed  Google Scholar 

  4. Kettenberger H, Armache K-J, Cramer P (2004) Complete RNA polymerase II elongation complex structure and its interactions with NTP and TFIIS. Mol Cell 16:955–965. https://doi.org/10.1016/j.molcel.2004.11.040

    CAS  CrossRef  PubMed  Google Scholar 

  5. Kettenberger H, Armache K-J, Cramer P (2003) Architecture of the RNA polymerase II-TFIIS complex and implications for mRNA cleavage. Cell 114:347–357. https://doi.org/10.1016/S0092-8674(03)00598-1

  6. Wang D, Bushnell DA, Huang X, Westover KD, Levitt M, Kornberg RD (2009) Structural basis of transcription: Backtracked RNA polymerase II at 3.4 Å resolution. Science 324:1203–1206. https://doi.org/10.1126/science.1168729

  7. Liu X, Bushnell DA, Wang D, Calero G, Kornberg RD (2010) Structure of an RNA polymerase II–TFIIB complex and the transcription initiation mechanism. Science 327:206. https://doi.org/10.1126/science.1182015

  8. Sainsbury S, Niesser J, Cramer P (2013) Structure and function of the initially transcribing RNA polymerase II-TFIIB complex. Nature 493:437–440. https://doi.org/10.1038/nature11715

    CAS  CrossRef  PubMed  Google Scholar 

  9. Sainsbury S, Bernecky C, Cramer P (2015) Structural basis of transcription initiation by RNA polymerase II. Nat Rev Mol Cell Biol 16:129–143. https://doi.org/10.1038/nrm3952

    CAS  CrossRef  PubMed  Google Scholar 

  10. McMullan G, Faruqi AR, Henderson R (2016) Direct electron detectors. Methods Enzymol 579:1–17. https://doi.org/10.1016/bs.mie.2016.05.056

    CAS  CrossRef  PubMed  Google Scholar 

  11. Scheres SHW (2012) A Bayesian view on cryo-EM structure determination. J Mol Biol 415:406–418. https://doi.org/10.1016/j.jmb.2011.11.010

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  12. Zivanov J, Nakane T, Forsberg B, Kimanius D, Hagen WJH, Lindahl E, Scheres SHW (2018) RELION-3: new tools for automated high-resolution cryo-EM structure determination. bioRxiv. https://doi.org/10.1101/421123

  13. Kühlbrandt W (2014) Biochemistry. The resolution revolution. Science 343:1443–1444. https://doi.org/10.1126/science.1251652

    CrossRef  PubMed  Google Scholar 

  14. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60:2126–2132. https://doi.org/10.1107/S0907444904019158

    CAS  CrossRef  PubMed  Google Scholar 

  15. Geiger SR, Lorenzen K, Schreieck A, Hanecker P, Kostrewa D, Heck AJR, Cramer P (2010) RNA polymerase I contains a TFIIF-related DNA-binding subcomplex. Mol Cell 39:583–594. https://doi.org/10.1016/j.molcel.2010.07.028

    CAS  CrossRef  PubMed  Google Scholar 

  16. Kuhn C-D, Geiger SR, Baumli S, Gartmann M, Gerber J, Jennebach S, Mielke T, Tschochner H, Beckmann R, Cramer P (2007) Functional architecture of RNA polymerase I. Cell 131:1260–1272. https://doi.org/10.1016/j.cell.2007.10.051

    CAS  CrossRef  PubMed  Google Scholar 

  17. Merkl PE, Pilsl M, Fremter T, Schwank K, Engel C, Längst G, Milkereit P, Griesenbeck J, Tschochner H (2020) RNA polymerase I (Pol I) passage through nucleosomes depends on Pol I subunits binding its lobe structure. J Biol Chem 295:4782–4795. https://doi.org/10.1074/jbc.RA119.011827

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  18. Jennebach S, Herzog F, Aebersold R, Cramer P (2012) Crosslinking-MS analysis reveals RNA polymerase I domain architecture and basis of rRNA cleavage. Nucleic Acids Res 40:5591–5601. https://doi.org/10.1093/nar/gks220

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  19. Lisica A, Engel C, Jahnel M, Roldán É, Galburt EA, Cramer P, Grill SW (2016) Mechanisms of backtrack recovery by RNA polymerases I and II. Proc Natl Acad Sci U S A 113:2946–2951. https://doi.org/10.1073/pnas.1517011113

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  20. Fath S, Kobor MS, Philippi A, Greenblatt J, Tschochner H (2004) Dephosphorylation of RNA polymerase I by Fcp1p is required for efficient rRNA synthesis. J Biol Chem 279:25251–25259. https://doi.org/10.1074/jbc.M401867200

    CAS  CrossRef  PubMed  Google Scholar 

  21. Clemente-Blanco A, Mayán-Santos M, Schneider DA, Machín F, Jarmuz A, Tschochner H, Aragón L (2009) Cdc14 inhibits transcription by RNA polymerase I during anaphase. Nature 458:219–222. https://doi.org/10.1038/nature07652

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  22. Mayer C, Zhao J, Yuan X, Grummt I (2004) mTOR-dependent activation of the transcription factor TIF-IA links rRNA synthesis to nutrient availability. Genes Dev 18:423–434. https://doi.org/10.1101/gad.285504

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  23. Hannan KM, Brandenburger Y, Jenkins A, Sharkey K, Cavanaugh A, Rothblum L, Moss T, Poortinga G, McArthur GA, Pearson RB, Hannan RD (2003) mTOR-dependent regulation of ribosomal gene transcription requires S6K1 and is mediated by phosphorylation of the carboxy-terminal activation domain of the nucleolar transcription factor UBF. Mol Cell Biol 23:8862–8877. https://doi.org/10.1128/mcb.23.23.8862-8877.2003

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  24. Cheung ACM, Cramer P (2012) A movie of RNA polymerase II transcription. Cell 149:1431–1437. https://doi.org/10.1016/j.cell.2012.06.006

    CAS  CrossRef  PubMed  Google Scholar 

  25. Moss T, Langlois F, Gagnon-Kugler T, Stefanovsky V (2007) A housekeeper with power of attorney: the rRNA genes in ribosome biogenesis. Cell Mol Life Sci 64:29–49. https://doi.org/10.1007/s00018-006-6278-1

    CAS  CrossRef  PubMed  Google Scholar 

  26. Goodfellow SJ, Zomerdijk JCBM (2013) Basic mechanisms in RNA polymerase I transcription of the ribosomal RNA genes. Subcell Biochem 61:211–236. https://doi.org/10.1007/978-94-007-4525-4_10

    CAS  CrossRef  PubMed  Google Scholar 

  27. Keener J, Josaitis CA, Dodd JA, Nomura M (1998) Reconstitution of yeast RNA polymerase I transcription in vitro from purified components. TATA-binding protein is not required for basal transcription. J Biol Chem 273:33795–33802

    CAS  CrossRef  Google Scholar 

  28. Milkereit P, Tschochner H (1998) A specialized form of RNA polymerase I, essential for initiation and growth-dependent regulation of rRNA synthesis, is disrupted during transcription. EMBO J 17:3692–3703. https://doi.org/10.1093/emboj/17.13.3692

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  29. Reiter A, Hamperl S, Seitz H, Merkl P, Perez-Fernandez J, Williams L, Gerber J, Németh A, Léger I, Gadal O, Milkereit P, Griesenbeck J, Tschochner H (2012) The Reb1-homologue Ydr026c/Nsi1 is required for efficient RNA polymerase I termination in yeast. EMBO J 31:3480–3493. https://doi.org/10.1038/emboj.2012.185

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  30. Merkl P, Perez-Fernandez J, Pilsl M, Reiter A, Williams L, Gerber J, Böhm M, Deutzmann R, Griesenbeck J, Milkereit P, Tschochner H (2014) Binding of the termination factor Nsi1 to its cognate DNA site is sufficient to terminate RNA polymerase I transcription in vitro and to induce termination in vivo. Mol Cell Biol 34:3817–3827. https://doi.org/10.1128/MCB.00395-14

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  31. Bischler N, Brino L, Carles C, Riva M, Tschochner H, Mallouh V, Schultz P (2002) Localization of the yeast RNA polymerase I-specific subunits. EMBO J 21:4136–4144. https://doi.org/10.1093/emboj/cdf392

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  32. Milkereit P, Schultz P, Tschochner H (1997) Resolution of RNA polymerase I into dimers and monomers and their function in transcription. Biol Chem 378:1433–1443

    CAS  CrossRef  Google Scholar 

  33. Engel C, Sainsbury S, Cheung AC, Kostrewa D, Cramer P (2013) RNA polymerase I structure and transcription regulation. Nature 502:650–655. https://doi.org/10.1038/nature12712

    CAS  CrossRef  PubMed  Google Scholar 

  34. Fernández-Tornero C, Moreno-Morcillo M, Rashid UJ, Taylor NMI, Ruiz FM, Gruene T, Legrand P, Steuerwald U, Müller CW (2013) Crystal structure of the 14-subunit RNA polymerase I. Nature 502:644–649. https://doi.org/10.1038/nature12636

    CAS  CrossRef  PubMed  Google Scholar 

  35. Kostrewa D, Kuhn C-D, Engel C, Cramer P (2015) An alternative RNA polymerase I structure reveals a dimer hinge. Acta Crystallogr D Biol Crystallogr 71:1850–1855. https://doi.org/10.1107/S1399004715012651

    CAS  CrossRef  PubMed  Google Scholar 

  36. Fernández-Tornero C (2018) RNA polymerase I activation and hibernation: unique mechanisms for unique genes. Transcription 9:248–254. https://doi.org/10.1080/21541264.2017.1416267

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  37. Torreira E, Louro JA, Pazos I, González-Polo N, Gil-Carton D, Duran AG, Tosi S, Gallego O, Calvo O, Fernández-Tornero C (2017) The dynamic assembly of distinct RNA polymerase I complexes modulates rDNA transcription. elife 6:e20832. https://doi.org/10.7554/eLife.20832

    CrossRef  PubMed  PubMed Central  Google Scholar 

  38. Hanske J, Sadian Y, Müller CW (2018) The cryo-EM resolution revolution and transcription complexes. Curr Opin Struct Biol 52:8–15. https://doi.org/10.1016/j.sbi.2018.07.002

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  39. Engel C, Neyer S, Cramer P (2018) Distinct mechanisms of transcription initiation by RNA polymerases I and II. Annu Rev Biophys 47:425–446. https://doi.org/10.1146/annurev-biophys-070317-033058

    CAS  CrossRef  PubMed  Google Scholar 

  40. Jochem L, Ramsay EP, Vannini A (2017) RNA polymerase I, bending the rules? EMBO J 36:2664–2666. https://doi.org/10.15252/embj.201797924

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  41. Jackobel AJ, Han Y, He Y, Knutson BA (2018) Breaking the mold: structures of the RNA polymerase I transcription complex reveal a new path for initiation. Transcription 9:255–261. https://doi.org/10.1080/21541264.2017.1416268

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  42. Blattner C, Jennebach S, Herzog F, Mayer A, Cheung ACM, Witte G, Lorenzen K, Hopfner K-P, Heck AJR, Aebersold R, Cramer P (2011) Molecular basis of Rrn3-regulated RNA polymerase I initiation and cell growth. Genes Dev 25:2093–2105. https://doi.org/10.1101/gad.17363311

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  43. Engel C, Plitzko J, Cramer P (2016) RNA polymerase I-Rrn3 complex at 4.8 Å resolution. Nat Commun 7:12129. https://doi.org/10.1038/ncomms12129

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  44. Pilsl M, Crucifix C, Papai G, Krupp F, Steinbauer R, Griesenbeck J, Milkereit P, Tschochner H, Schultz P (2016) Structure of the initiation-competent RNA polymerase I and its implication for transcription. Nat Commun 7:12126. https://doi.org/10.1038/ncomms12126

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  45. Knutson BA, Hahn S (2011) Yeast Rrn7 and human TAF1B are TFIIB-related RNA polymerase I general transcription factors. Science 333:1637–1640. https://doi.org/10.1126/science.1207699

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  46. Naidu S, Friedrich JK, Russell J, Zomerdijk JCBM (2011) TAF1B is a TFIIB-like component of the basal transcription machinery for RNA polymerase I. Science 333:1640–1642. https://doi.org/10.1126/science.1207656

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  47. Heiss FB, Daiß JL, Becker P, Engel C (2021) Conserved strategies of RNA polymerase I hibernation and activation. Nat Commun 12:758. https://doi.org/10.1038/s41467-021-21031-8

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  48. Engel C, Gubbey T, Neyer S, Sainsbury S, Oberthuer C, Baejen C, Bernecky C, Cramer P (2017) Structural basis of RNA polymerase I transcription initiation. Cell 169:120–131.e22. https://doi.org/10.1016/j.cell.2017.03.003

    CAS  CrossRef  PubMed  Google Scholar 

  49. Han Y, Yan C, Nguyen THD, Jackobel AJ, Ivanov I, Knutson BA, He Y (2017) Structural mechanism of ATP-independent transcription initiation by RNA polymerase I. elife 6:e27414. https://doi.org/10.7554/eLife.27414

    CrossRef  PubMed  PubMed Central  Google Scholar 

  50. Sadian Y, Tafur L, Kosinski J, Jakobi AJ, Wetzel R, Buczak K, Hagen WJ, Beck M, Sachse C, Müller CW (2017) Structural insights into transcription initiation by yeast RNA polymerase I. EMBO J 36:2698–2709. https://doi.org/10.15252/embj.201796958

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  51. Bier M, Fath S, Tschochner H (2004) The composition of the RNA polymerase I transcription machinery switches from initiation to elongation mode. FEBS Lett 564:41–46. https://doi.org/10.1016/S0014-5793(04)00311-4

    CAS  CrossRef  PubMed  Google Scholar 

  52. Herdman C, Mars J-C, Stefanovsky VY, Tremblay MG, Sabourin-Felix M, Lindsay H, Robinson MD, Moss T (2017) A unique enhancer boundary complex on the mouse ribosomal RNA genes persists after loss of Rrn3 or UBF and the inactivation of RNA polymerase I transcription. PLoS Genet 13:e1006899. https://doi.org/10.1371/journal.pgen.1006899

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  53. Sadian Y, Baudin F, Tafur L, Murciano B, Wetzel R, Weis F, Müller CW (2019) Molecular insight into RNA polymerase I promoter recognition and promoter melting. Nat Commun 10:5543. https://doi.org/10.1038/s41467-019-13510-w

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  54. Pilsl M, Engel C (2020) Structural basis of RNA polymerase I pre-initiation complex formation and promoter melting. Nat Commun 11:1206. https://doi.org/10.1038/s41467-020-15052-y

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  55. Neyer S, Kunz M, Geiss C, Hantsche M, Hodirnau V-V, Seybert A, Engel C, Scheffer MP, Cramer P, Frangakis AS (2016) Structure of RNA polymerase I transcribing ribosomal DNA genes. Nature 540(7634):607–610. https://doi.org/10.1038/nature20561

    CAS  CrossRef  PubMed  Google Scholar 

  56. Tafur L, Sadian Y, Hoffmann NA, Jakobi AJ, Wetzel R, Hagen WJH, Sachse C, Müller CW (2016) Molecular structures of transcribing RNA polymerase I. Mol Cell 64:1135–1143. https://doi.org/10.1016/j.molcel.2016.11.013

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  57. Beckouet F, Labarre-Mariotte S, Albert B, Imazawa Y, Werner M, Gadal O, Nogi Y, Thuriaux P (2008) Two RNA polymerase I subunits control the binding and release of Rrn3 during transcription. Mol Cell Biol 28:1596–1605. https://doi.org/10.1128/MCB.01464-07

    CAS  CrossRef  PubMed  Google Scholar 

  58. Tafur L, Sadian Y, Hanske J, Wetzel R, Weis F, Müller CW (2019) The cryo-EM structure of a 12-subunit variant of RNA polymerase I reveals dissociation of the A49-A34.5 heterodimer and rearrangement of subunit A12.2. elife 8:e43204. https://doi.org/10.7554/eLife.43204

    CrossRef  PubMed  PubMed Central  Google Scholar 

  59. Hoffmann NA, Jakobi AJ, Moreno-Morcillo M, Glatt S, Kosinski J, Hagen WJH, Sachse C, Müller CW (2015) Molecular structures of unbound and transcribing RNA polymerase III. Nature 528:231–236. https://doi.org/10.1038/nature16143

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  60. Sanz-Murillo M, Xu J, Belogurov GA, Calvo O, Gil-Carton D, Moreno-Morcillo M, Wang D, Fernández-Tornero C (2018) Structural basis of RNA polymerase I stalling at UV light-induced DNA damage. Proc Natl Acad Sci U S A 115:8972–8977. https://doi.org/10.1073/pnas.1802626115

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  61. Miller OL, Beatty BR (1969) Visualization of nucleolar genes. Science 164:955–957. https://doi.org/10.1126/science.164.3882.955

    CrossRef  PubMed  Google Scholar 

  62. Kramm K, Engel C, Grohmann D (2019) Transcription initiation factor TBP: old friend new questions. Biochem Soc Trans 47:411–423. https://doi.org/10.1042/BST20180623

    CAS  CrossRef  PubMed  Google Scholar 

  63. Bedwell GJ, Appling FD, Anderson SJ, Schneider DA (2012) Efficient transcription by RNA polymerase I using recombinant core factor. Gene 492:94–99. https://doi.org/10.1016/j.gene.2011.10.049

    CAS  CrossRef  PubMed  Google Scholar 

  64. Schilbach S, Hantsche M, Tegunov D, Dienemann C, Wigge C, Urlaub H, Cramer P (2017) Structures of transcription pre-initiation complex with TFIIH and mediator. Nature 551:204–209. https://doi.org/10.1038/nature24282

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  65. He Y, Yan C, Fang J, Inouye C, Tjian R, Ivanov I, Nogales E (2016) Near-atomic resolution visualization of human transcription promoter opening. Nature 533:359–365. https://doi.org/10.1038/nature17970

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  66. Abascal-Palacios G, Ramsay EP, Beuron F, Morris E, Vannini A (2018) Structural basis of RNA polymerase III transcription initiation. Nature 553:301–306. https://doi.org/10.1038/nature25441

    CAS  CrossRef  PubMed  Google Scholar 

  67. Vorländer MK, Khatter H, Wetzel R, Hagen WJH, Müller CW (2018) Molecular mechanism of promoter opening by RNA polymerase III. Nature 553:295–300. https://doi.org/10.1038/nature25440

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  68. Ramsay EP, Abascal-Palacios G, Daiß JL, King H, Gouge J, Pilsl M, Beuron F, Morris E, Gunkel P, Engel C, Vannini A (2020) Structure of human RNA polymerase III. Nat Commun 11:6409. https://doi.org/10.1038/s41467-020-20262-5

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  69. Shukla AK, Westfield GH, Xiao K, Reis RI, Huang L-Y, Tripathi-Shukla P, Qian J, Li S, Blanc A, Oleskie AN, Dosey AM, Su M, Liang C-R, Gu L-L, Shan J-M, Chen X, Hanna R, Choi M, Yao XJ, Klink BU, Kahsai AW, Sidhu SS, Koide S, Penczek PA, Kossiakoff AA, Woods VL, Kobilka BK, Skiniotis G, Lefkowitz RJ (2014) Visualization of arrestin recruitment by a G-protein-coupled receptor. Nature 512:218–222. https://doi.org/10.1038/nature13430

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  70. Kramm K, Schröder T, Gouge J, Vera AM, Gupta K, Heiss FB, Liedl T, Engel C, Berger I, Vannini A, Tinnefeld P, Grohmann D (2020) DNA origami-based single-molecule force spectroscopy elucidates RNA polymerase III pre-initiation complex stability. Nat Commun 11:2828. https://doi.org/10.1038/s41467-020-16702-x

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  71. Leitner A, Faini M, Stengel F, Aebersold R (2016) Crosslinking and mass spectrometry: an integrated technology to understand the structure and function of molecular machines. Trends Biochem Sci 41:20–32. https://doi.org/10.1016/j.tibs.2015.10.008

    CAS  CrossRef  PubMed  Google Scholar 

  72. Schmidt C, Urlaub H (2017) Combining cryo-electron microscopy (cryo-EM) and cross-linking mass spectrometry (CX-MS) for structural elucidation of large protein assemblies. Curr Opin Struct Biol 46:157–168. https://doi.org/10.1016/j.sbi.2017.10.005

    CAS  CrossRef  PubMed  Google Scholar 

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Acknowledgements

The authors thank all members of the Structural Biochemistry group and Biochemistry I/III departments of the University of Regensburg for help and discussions. This work was supported by the Emmy-Noether-Programme (DFG grant no. EN 1204/1-1 to CE) and SFB 960 TP-A8.

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Pilsl, M., Engel, C. (2022). Structural Studies of Eukaryotic RNA Polymerase I Using Cryo-Electron Microscopy. In: Entian, KD. (eds) Ribosome Biogenesis. Methods in Molecular Biology, vol 2533. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2501-9_5

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