Abstract
Messenger RNA synthesis (mRNA) accounts for a small fraction of total RNA synthesis in growing eukaryotic cells. The bulk of cellular transcription is devoted to ribosomal RNA (rRNA) synthesis (Warner, Trends Biochem Sci 24:437–440, 1999). Several unique characteristics of the rDNA and RNA polymerase I must be considered in order to accurately quantify the synthesis rate of rRNA or to characterize its processing. Indeed, an entirely different set of techniques must be applied to the study of rRNA synthesis than is routinely to study mRNA synthesis. Five of the most useful strategies for genetic and molecular analysis of rRNA synthesis and regulation are outlined in this chapter. The techniques described were developed for characterization of the model eukaryote Saccharomyces cerevisiae; however, many of these strategies can be adapted for studies in other eukaryotic cells.
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References
Larson DR, Zenklusen D, Wu B et al (2011) Real-time observation of transcription initiation and elongation on an endogenous yeast gene. Science 332:475–478
Kobayashi T, Heck DJ, Nomura M et al (1998) Expansion and contraction of ribosomal DNA repeats in Saccharomyces cerevisiae: requirement of replication fork blocking (Fob1) protein and the role of RNA polymerase I. Genes Dev 12:3821–3830
Clemente-Blanco A, Mayan-Santos M, Schneider DA et al (2009) Cdc14 inhibits transcription by RNA polymerase I during anaphase. Nature 458:219–222
Anderson SJ, Sikes ML, Zhang Y et al (2011) The transcription elongation factor Spt5 influences transcription by RNA polymerase I positively and negatively. J Biol Chem 286: 18816–18824
Zhang Y, Smith AD IV, Renfrow MB et al (2010) The RNA polymerase-associated factor 1 complex (Paf1C) directly increases the elongation rate of RNA polymerase I and is required for efficient regulation of rRNA synthesis. J Biol Chem 285:14152–14159
Schneider DA, French SL, Osheim YN et al (2006) RNA polymerase II elongation factors Spt4p and Spt5p play roles in transcription elongation by RNA polymerase I and rRNA processing. Proc Natl Acad Sci U S A 103: 12707–12712
Conconi A, Widmer RM, Koller T et al (1989) Two different chromatin structures coexist in ribosomal RNA genes throughout the cell cycle. Cell 57:753–761
Dammann R, Lucchini R, Koller T et al (1993) Chromatin structures and transcription of rDNA in yeast Saccharomyces cerevisiae. Nucleic Acids Res 21:2331–2338
Sandmeier JJ, French SL, Osheim YN et al (2002) RPD3 is required for the inactivation of yeast ribosomal DNA genes in stationary phase. EMBO J 21:4959–4968
Nogi Y, Vu L, Nomura M (1991) An approach for isolation of mutants defective in 35S ribosomal RNA synthesis in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 88:7026–7030
Nogi Y, Yano R, Nomura M (1991) Synthesis of large rRNAs by RNA polymerase II in mutants of Saccharomyces cerevisiae defective in RNA polymerase I. Proc Natl Acad Sci U S A 88:3962–3966
Hontz RD, Niederer RO, Johnson JM et al (2009) Genetic identification of factors that modulate ribosomal DNA transcription in Saccharomyces cerevisiae. Genetics 182:105–119
Osheim YN, French SL, Sikes ML et al (2009) Electron microscope visualization of RNA transcription and processing in Saccharomyces cerevisiae by Miller chromatin spreading. Methods Mol Biol 464:55–69
Warner JR (1999) The economics of ribosome biosynthesis in yeast. Trends Biochem Sci 24: 437–440
Cioci F, Vu L, Eliason K et al (2003) Silencing in yeast rDNA chromatin: reciprocal relationship in gene expression between RNA polymerase I and II. Mol Cell 12:135–145
Schneider DA, Michel A, Sikes ML et al (2007) Transcription elongation by RNA polymerase I is linked to efficient rRNA processing and ribosome assembly. Mol Cell 26:217–229
Hontz RD, French SL, Oakes ML et al (2008) Transcription of multiple yeast ribosomal DNA genes requires targeting of UAF to the promoter by Uaf30. Mol Cell Biol 28:6709–6719
Oakes M, Aris JP, Brockenbrough JS et al (1998) Mutational analysis of the structure and localization of the nucleolus in the yeast Saccharomyces cerevisiae. J Cell Biol 143:23–34
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Johnson, J.M., Smith, J.S., Schneider, D.A. (2014). A User’s Guide to the Ribosomal DNA in Saccharomyces cerevisiae . In: Smith, J., Burke, D. (eds) Yeast Genetics. Methods in Molecular Biology, vol 1205. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1363-3_18
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DOI: https://doi.org/10.1007/978-1-4939-1363-3_18
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