The large subunit of RNA polymerase II is a substrate of the Rsp5 ubiquitin-protein ligase.

The E3 ubiquitin-protein ligases play an important role in controlling substrate specificity of the ubiquitin proteolysis system. A biochemical approach was taken to identify substrates of Rsp5, an essential hect (homologous to E6-AP carboxyl terminus) E3 of Saccharomyces cerevisiae. We show here that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II (Rpb1) in vitro. Stable complex formation between Rsp5 and Rpb1 was also detected in yeast cell extracts, and repression of RSP5 expression in vivo led to an elevated steady-state level of Rpb1. The amino-terminal domain of Rsp5 mediates binding to Rpb1, while the carboxyl-terminal domain of Rpb1, containing the heptapeptide repeats characteristic of polymerase II, is necessary and sufficient for binding to Rsp5. Fusion of the Rpb1 carboxyl-terminal domain to another protein also causes that protein to be ubiquitinated by Rsp5. These findings indicate that Rsp5 targets at least a subset of cellular Rpb1 molecules for ubiquitin-dependent degradation and may therefore play a role in regulating polymerase II activities. In addition, the results support a model for hect E3 function in which the amino-terminal domain mediates substrate binding, while the carboxyl-terminal hect domain catalyzes ubiquitination of bound substrates.

[1]  S. Kumar,et al.  Identification of a set of genes with developmentally down-regulated expression in the mouse brain. , 1992, Biochemical and biophysical research communications.

[2]  M. Imhof,et al.  Yeast RSP5 and its human homolog hRPF1 potentiate hormone-dependent activation of transcription by human progesterone and glucocorticoid receptors , 1996, Molecular and cellular biology.

[3]  D. Beach,et al.  Pub1 acts as an E6‐AP‐like protein ubiquitiin ligase in the degradation of cdc25. , 1996, The EMBO journal.

[4]  H. Yashiroda,et al.  Bul1, a new protein that binds to the Rsp5 ubiquitin ligase in Saccharomyces cerevisiae , 1996, Molecular and Cellular Biology.

[5]  R. Vierstra,et al.  Cloning of ubiquitin activating enzyme from wheat and expression of a functional protein in Escherichia coli. , 1990, The Journal of biological chemistry.

[6]  M. Hochstrasser,et al.  Protein Degradation or Regulation: Ub the Judge , 1996, Cell.

[7]  M. Kirschner,et al.  A 20s complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B , 1995, Cell.

[8]  M. Scheffner,et al.  The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53 , 1993, Cell.

[9]  A. Hershko,et al.  The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis. , 1995, Molecular biology of the cell.

[10]  M. Scheffner,et al.  A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[11]  B. André,et al.  NPI1, an essential yeast gene involved in induced degradation of Gap1 and Fur4 permeases, encodes the Rsp5 ubiquitin—protein ligase , 1995, Molecular microbiology.

[12]  M. Sullivan,et al.  Homologs of the essential ubiquitin conjugating enzymes UBC1, 4, and 5 in yeast are encoded by a multigene family in Arabidopsis thaliana. , 1993, The Plant journal : for cell and molecular biology.

[13]  R. Halaban,et al.  UV-induced ubiquitination of RNA polymerase II: a novel modification deficient in Cockayne syndrome cells. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[14]  C. Ingles,et al.  The C-terminal domain of the largest subunit of RNA polymerase II of Saccharomyces cerevisiae, Drosophila melanogaster, and mammals: a conserved structure with an essential function , 1988, Molecular and cellular biology.

[15]  J. W. Rooney,et al.  SPT3 interacts with TFIID to allow normal transcription in Saccharomyces cerevisiae. , 1992, Genes & development.

[16]  Martin Scheffner,et al.  Protein ubiquitination involving an E1–E2–E3 enzyme ubiquitin thioester cascade , 1995, Nature.

[17]  Aaron Ciechanover,et al.  The ubiquitin-proteasome proteolytic pathway , 1994, Cell.

[18]  K. Davies,et al.  Coiled-coil regions in the carboxy-terminal domains of dystrophin and related proteins: potentials for protein-protein interactions. , 1995, Trends in biochemical sciences.

[19]  J. Lis,et al.  Phosphorylation of RNA polymerase II C-terminal domain and transcriptional elongation , 1994, Nature.

[20]  M. Scheffner,et al.  Localization of the E6-AP regions that direct human papillomavirus E6 binding, association with p53, and ubiquitination of associated proteins , 1993, Molecular and cellular biology.

[21]  J. D. Clark,et al.  A novel arachidonic acid-selective cytosolic PLA2 contains a Ca2+-dependent translocation domain with homology to PKC and GAP , 1991, Cell.

[22]  M. Scheffner,et al.  Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53 , 1993, Molecular and cellular biology.

[23]  A. Varshavsky,et al.  The recognition component of the N‐end rule pathway. , 1990, The EMBO journal.

[24]  D. Reinberg,et al.  Specific interaction between the nonphosphorylated form of RNA polymerase II and the TATA-binding protein , 1992, Cell.

[25]  P. Silver,et al.  Mutations that alter both localization and production of a yeast nuclear protein. , 1988, Genes & development.

[26]  P. Bork,et al.  Characterization of a novel protein‐binding module — the WW domain , 1995, FEBS letters.

[27]  O. Staub,et al.  WW domains of Nedd4 bind to the proline‐rich PY motifs in the epithelial Na+ channel deleted in Liddle's syndrome. , 1996, The EMBO journal.