Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2000 May 1;149(3):575–590. doi: 10.1083/jcb.149.3.575

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2000 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

PMC Copyright notice

The nucleolar assembly isolated from pol I–deficient cells can recruit pol I to result in a transcriptionally active complex. (A) The nucleolar components copurify and coprecipitate when prepared from yeast strains not active in pol I–dependent transcription. (Upper panel) 8 μg of fraction PA600 derived from stationary cells (lane 3), from strain delta 135, which was disrupted in the A135 pol I subunit (strain OG39-6d) (lane 2), and from growing cells was analyzed by Western blotting with antibodies as indicated. (Lower panel) 1.25 μg of each PA600 fraction was analyzed in promoter-dependent transcription. (B) Purified pol I can be reassociated into a functionable assembly. Purified pol I-p (7 μl), which also contained pol I/Rrn3p complex but lacked TBP-cpl and fraction B600 (see Milkereit et al. 1997; Milkereit and Tschochner 1998) and thus was not able to initiate transcription on the rDNA promoter by itself, was combined with 12.5 μg (lanes 1–3) or without (lanes 4–6) fraction PA600 derived from strain Δ135 lacking A135 and adjusted to 1.5 M potassium acetate. The fractions were dialyzed against buffer BU100 containing 100 mM potassium acetate and centrifuged. Precipitated components were resolubilized in buffer BU600 containing 600 mM acetate and analyzed in promoter-dependent transcription. (C) In vitro assay to analyze the pol I–recruiting activity. Purified pol I coprecipitates with components present in fraction B2000 (Δ135) rather than with constituents in fraction B600 (Δ135). 0.5 μg pol I-A was combined with either 6 μg fraction PA600, 4.8 μg fraction B600, or fraction B2000, all derived from the pol I–deficient strain Δ135 in a total volume of 25 μl, adjusted to 1.5 M potassium acetate, dialyzed against buffer BU100, and centrifuged. Precipitates were resolubilized in 15 μl BU600 and analyzed in nonspecific RNA synthesis.