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© The Rockefeller University Press, 0021-9525/2000//575 $5.00
The Journal of Cell Biology, Volume 149, Number 3, , 2000 575-590

Original Article

Association of Yeast RNA Polymerase I with a Nucleolar Substructure Active in Rrna Synthesis and Processing



Stephan Fatha, Philipp Milkereita, Alexandre V. Podtelejnikovb, Nicolas Bischlerc, Patrick Schultzc, Mirko Biera, Matthias Mannb, and Herbert Tschochnera

a Biochemie-Zentrum Heidelberg, D-69120 Heidelberg, Germany
b Protein Interaction Laboratory, Odense University, DK 5230 Odense Middle, Denmark
c Institut de Génétique et de Biologie Moléculaire et Cellulaire, F-67404 Illkirch Cedex, France
Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany.49-6221-54436649-6221-544155

im4{at}ix.urz.uni-heidelberg.de

A novel ribonucleoprotein complex enriched in nucleolar proteins was purified from yeast extracts and constituents were identified by mass spectrometry. When isolated from rapidly growing cells, the assembly contained ribonucleic acid (RNA) polymerase (pol) I, and some of its transcription factors like TATA-binding protein (TBP), Rrn3p, Rrn5p, Rrn7p, and Reb1p along with rRNA processing factors, like Nop1p, Cbf5p, Nhp2p, and Rrp5p. The small nucleolar RNAs (snoRNAs) U3, U14, and MRP were also found to be associated with the complex, which supports accurate transcription, termination, and pseudouridylation of rRNA. Formation of the complex did not depend on pol I, and the complex could efficiently recruit exogenous pol I into active ribosomal DNA (rDNA) transcription units. Visualization of the complex by electron microscopy and immunogold labeling revealed a characteristic cluster-forming network of nonuniform size containing nucleolar proteins like Nop1p and Fpr3p and attached pol I. Our results support the idea that a functional nucleolar subdomain formed independently of the state of rDNA transcription may serve as a scaffold for coordinated rRNA synthesis and processing.

Key Words: in vitro transcription • Saccharomyces cerevisiae • ribosome biogenesis • matrix assisted laser desorption ionization (MALDI) mass spectrometry • nucleolus

© 2000 The Rockefeller University Press

Abbreviations used in this paper: CF, core factor; HA, hemagglutinin; MALDI, matrix assisted laser desorption ionization; pol, RNA polymerase; rDNA, ribosomal DNA; snoRNA, small nucleolar RNA; TBP, TATA-binding protein; UAF, upstream activating factor; WCE, whole cell extract.


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