Biochemical and genetic characterization of the Paf1/RNA polymerase
II complex.
Cherie L. Mueller (1), Judith A. Jaehning (2)
(1) Department of Biochemistry and Molecular Genetics;
(2) Molecular Biology Program, University of Colorado HSC, 4200 E. 9th
Ave., Denver, CO 80262, USA
The yeast Paf1/RNA polymerase II (pol II) complex is biochemically and
functionally distinct from the Srb-mediator form of pol II holoenzyme,
and is required for full expression of a subset of genes including many
of those whose expression is regulated during the cell cycle, including
CLN1, HO and RNR1. Using Tandem Affinity
Purification (TAP) tags and mass-spectrometry we isolated the Paf1
complex and identified additional components including Ctr9, Rtf1 and
Leo1. paf1 and ctr9 strains have very similar pleiotropic
phenotypes, which are unaltered when the two mutations are combined. In
contrast, deletion of LEO1 or RTF1 leads to few obvious
phenotypes, but combining leo1 or rtf1 with paf1
leads to suppression of many paf1 phenotypes including slow
growth, and sensitivity to high temperature, caffeine and hydroxyurea.
In addition, expression of several genes whose expression is reduced in
paf1 is restored to wild type levels in the rtf1 paf1
double mutant. Using chromatin immuno-precipitation, we have found that
Paf1 complex component Leo1 can be detected throughout the length of the
CLN1 and HO genes in wild type cells. In a paf1
mutant the Leo1 signal is dramatically reduced in concert with the
reduced expression of the genes. We are currently testing the hypothesis
that lack of Paf1 results in a defective complex and a block in
transcription, which is relieved by removal of Leo1 or Rtf1.
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