title: Analysis of S. cerevisiae RNA Polymerase I Transcription In Vitro.
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.
authors: Biochemie-Zentrum Regensburg (BZR), Lehrstuhl Biochemie III, Universität Regensburg, 93053, Regensburg, Germany.
authors: Michael
authors: M
authors: Pilsl
authors: Biochemie-Zentrum Regensburg (BZR), Lehrstuhl Biochemie III, Universität Regensburg, 93053, Regensburg, Germany.|Department of Microbiology and Immunobiology, Harvard Medical School, New Research Building Room 954, 77 Avenue Louis Pasteur, Boston, MA, 02115, USA.
authors: Philipp E
authors: PE
authors: Merkl
authors: Biochemie-Zentrum Regensburg (BZR), Lehrstuhl Biochemie III, Universität Regensburg, 93053, Regensburg, Germany.
authors: Philipp
authors: P
authors: Milkereit
authors: Biochemie-Zentrum Regensburg (BZR), Lehrstuhl Biochemie III, Universität Regensburg, 93053, Regensburg, Germany.
authors: Joachim
authors: J
authors: Griesenbeck
authors: Biochemie-Zentrum Regensburg (BZR), Lehrstuhl Biochemie III, Universität Regensburg, 93053, Regensburg, Germany. herbert.tschochner@ur.de.
authors: Herbert
authors: H
authors: Tschochner
mesh_terms: D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D066298:In Vitro Techniques; D035463:Pol1 Transcription Initiation Complex Proteins / Q000302:isolation & purification / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012318:RNA Polymerase I / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000235:genetics; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic
chemical_list: D004268:DNA-Binding Proteins; C574300:Nsi1 protein, S cerevisiae; D035463:Pol1 Transcription Initiation Complex Proteins; D012335:RNA, Ribosomal; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D012318:RNA Polymerase I
keywords: In vitro transcription; RNA polymerase I; Saccharomyces cerevisiae