Saccharomyces cerevisiae RNA Polymerase II Is Affected by Kluyveromyces lactis Zymocin

Daniel Jablonowski,Raffael Schaffrath

doi:10.1074/jbc.m203354200

Daniel Jablonowski, Raffael Schaffrath

Open Access

https://doi.org/10.1074/jbc.m203354200

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Abstract

The G(1) arrest imposed by Kluyveromyces lactis zymocin on Saccharomyces cerevisiae cells requires a functional RNA polymerase II (pol II) Elongator complex. In studying a link between zymocin and pol II, progressively truncating the carboxyl-terminal domain (CTD) of pol II was found to result in zymocin hypersensitivity as did mutations in four different CTD kinase genes. Consistent with the notion that Elongator preferentially associates with hyperphosphorylated (II0) rather than hypophosphorylated (IIA) pol II, the II0/IIA ratio was imbalanced toward II0 on zymocin treatment and suggests zymocin affects pol II function, presumably in an Elongator-dependent manner. As judged from chromatin immunoprecipitations, zymocin-arrested cells were affected with regards to pol II binding to the ADH1 promotor and pol II transcription of the ADH1 gene. Thus, zymocin may interfere with pol II recycling, a scenario assumed to lead to down-regulation of pol II transcription and eventually causing the observed G(1) arrest.

Highlights

Yeast killer toxins are genetically and biochemically diverse
Genetic scenarios leading to reduced polymerase II (pol II) function by mutating individual pol II carboxyl-terminal domain (CTD)-cyclin-dependent kinases (CDKs) genes or by progressively truncating the CTD itself were found to confer zymocin hypersensitivity
Presumed stability of the CTD of the pol II subunit in these mutants was not affected, but rather assembly of pol II holoenzyme was interfered with so that its function was compromised with respect to interaction with mediator or with Elongator; both scenarios, CTD truncation and CTD-CDK mutation, would lead to a decline of pol II activity

Summary

EXPERIMENTAL PROCEDURES

Zymocin sensitivity or resistance was assessed using the killer eclipse assay [31] and zymocin YPD plate assays The latter involved partial purification of zymocin from AWJ137 cell-free culture supernatants and supplementation to YPD plates [5, 23]. Zymocin arrest of S. cerevisiae strain LS20 involved partial purification of zymocin from K. lactis killer AWJ137 grown in YPD flask cultures for 48 h at 30 °C. After centrifugation (6,000 rpm) 50 ml of cell-free supernatants were mixed with 50 ml of 2ϫ YPD medium and inoculated with LS20 cells. These were arrested by zymocin on further incubating for 3– 6 h at 30 °C. Protein loadings were compared using a polyclonal rabbit antibody directed against the ␣- and ␤-subunits of yeast Pfk1p [33]. pol II quantitation used the Image Master 2D (Amersham Biosciences) program

Zymocin Affects RNA pol II Function

RESULTS

DISCUSSION

Daniel Jablonowski and Raffael Schaffrath