Abstract
RNA polymerase I (Pol I) activity is crucial to provide cells with sufficient amounts of ribosomal RNA (rRNA). Synthesis of rRNA takes place in the nucleolus, is tightly regulated and is coordinated with synthesis and assembly of ribosomal proteins, finally resulting in the formation of mature ribosomes. Many studies on Pol I mechanisms and regulation in the model organism S. cerevisiae were performed using either complex in vitro systems reconstituted from more or less purified fractions or genetic analyses. While providing many valuable insights these strategies did not always discriminate between direct and indirect effects in transcription initiation and termination, when mutated forms of Pol I subunits or transcription factors were investigated. Therefore, a well-defined minimal system was developed which allows to reconstitute highly efficient promoter-dependent Pol I initiation and termination of transcription. Transcription can be initiated at a minimal promoter only in the presence of recombinant core factor and extensively purified initiation competent Pol I. Addition of recombinant termination factors triggers transcriptional pausing and release of the ternary transcription complex. This minimal system represents a valuable tool to investigate the direct impact of (lethal) mutations in components of the initiation and termination complexes on the mechanism and regulation of rRNA synthesis.
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References
Lang WH, Morrow BE, Ju Q et al (1994) A model for transcription termination by RNA polymerase I. Cell 79(3):527–534
Schultz MC, Choe SY, Reeder RH (1991) Specific initiation by RNA polymerase I in a whole-cell extract from yeast. Proc Natl Acad Sci U S A 88(3):1004–1008
Keener J, Josaitis C, Dodd J et al (1998) Reconstitution of yeast RNA polymerase I transcription in vitro from purified factors. J Biol Chem 273:33795–33802
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
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
Bedwell GJ, Appling FD, Anderson SJ et al (2012) Efficient transcription by RNA polymerase I using recombinant core factor. Gene 492(1):94–99
Merkl P, Perez-Fernandez J, Pilsl M et al (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(20):3817–3827
Jeong SW, Lang WH, Reeder RH (1995) The release element of the yeast polymerase I transcription terminator can function independently of Reb1p. Mol Cell Biol 15(11):5929–5936
Lang WH, Reeder RH (1995) Transcription termination of RNA polymerase I due to a T-rich element interacting with Reb1p. Proc Natl Acad Sci U S A 92(21):9781–9785
Kadesch TR, Chamberlin MJ (1982) Studies of in vitro transcription by calf thymus RNA polymerase II using a novel duplex DNA template. J Biol Chem 257(9):5286–5295
Kuhn CD, Geiger SR, Baumli S et al (2007) Functional architecture of RNA polymerase I. Cell 131(7):1260–1272
Geiger SR, Lorenzen K, Schreieck A et al (2010) RNA polymerase I contains a TFIIF-related DNA-binding subcomplex. Mol Cell 39(4):583–594
Hamperl S, Brown CR, Perez-Fernandez J et al (2014) Purification of specific chromatin domains from single-copy gene loci in Saccharomyces cerevisiae. Methods Mol Biol 1094:329–341
Acknowledgement
This work was supported by grants of the DFG (SFB 960). P. E. M. was partly supported by a fellowship of the German National Academic Foundation.
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Pilsl, M., Merkl, P.E., Milkereit, P., Griesenbeck, J., Tschochner, H. (2016). Analysis of S. cerevisiae RNA Polymerase I Transcription In Vitro. In: Németh, A. (eds) The Nucleolus. Methods in Molecular Biology, vol 1455. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3792-9_8
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DOI: https://doi.org/10.1007/978-1-4939-3792-9_8
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