Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

RSP5, an essential gene of Saccharomyces cerevisiae, encodes a hect domain E3 ubiquitin-protein ligase. Hect E3 proteins have been proposed to consist of two broad functional domains: a conserved catalytic carboxyl-terminal domain of approximately 350 amino acids (the hect domain) and a large, nonconserved amino-terminal domain containing determinants of substrate specificity. We report here the mapping of the minimal region of Rsp5 necessary for its essential in vivo function, the minimal region necessary to stably interact with a substrate of Rsp5 (Rpb1, the large subunit of RNA polymerase II), and the finding that the hect domain, by itself, is sufficient for formation of the ubiquitin-thioester intermediate. Mutations within the hect domain that affect either the ability to form a ubiquitin-thioester or to catalyze substrate ubiquitination abrogate in vivo function, strongly suggesting that the ubiquitin-protein ligase activity of Rsp5 is intrinsically linked to its essential function. The amino-terminal region of Rsp5 contains three WW domains and a C2 calcium-binding domain. Two of the three WW domains are required for the essential in vivo function, while the C2 domain is not, and requirements for Rpb1 binding and ubiquitination lie within the region required for in vivo function. Together, these results support the two-domain model for hect E3 function and indicate that the WW domains play a role in the recognition of at least some of the substrates of Rsp5, including those related to its essential function. In addition, we show that haploid yeast strains bearing complete disruptions of either of two other hect E3 genes of yeast, designated HUL4 (YJR036C) and HUL5 (YGL141W), are viable.
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PMID:Functional domains of the Rsp5 ubiquitin-protein ligase. 985 58

Rsp5 is an E3 ubiquitin-protein ligase of Saccharomyces cerevisiae that belongs to the hect domain family of E3 proteins. We have previously shown that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II, Rpb1, in vitro. We show here that Rpb1 ubiquitination and degradation are induced in vivo by UV irradiation and by the UV-mimetic compound 4-nitroquinoline-1-oxide (4-NQO) and that a functional RSP5 gene product is required for this effect. The 26S proteasome is also required; a mutation of SEN3/RPN2 (sen3-1), which encodes an essential regulatory subunit of the 26S proteasome, partially blocks 4-NQO-induced degradation of Rpb1. These results suggest that Rsp5-mediated ubiquitination and degradation of Rpb1 are components of the response to DNA damage. A human WW domain-containing hect (WW-hect) E3 protein closely related to Rsp5, Rpf1/hNedd4, also binds and ubiquitinates both yeast and human Rpb1 in vitro, suggesting that Rpf1 and/or another WW-hect E3 protein mediates UV-induced degradation of the large subunit of polymerase II in human cells.
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PMID:Rsp5 ubiquitin-protein ligase mediates DNA damage-induced degradation of the large subunit of RNA polymerase II in Saccharomyces cerevisiae. 1049 Jun 34

RSP5 is an essential gene in Saccharomyces cerevisiae and was recently shown to form a physical and functional complex with RNA polymerase II (RNA pol II). The amino-terminal half of Rsp5 consists of four domains: a C2 domain, which binds membrane phospholipids; and three WW domains, which are protein interaction modules that bind proline-rich ligands. The carboxyl-terminal half of Rsp5 contains a HECT (homologous to E6-AP carboxyl terminus) domain that catalytically ligates ubiquitin to proteins and functionally classifies Rsp5 as an E3 ubiquitin-protein ligase. The C2 and WW domains are presumed to act as membrane localization and substrate recognition modules, respectively. We report that the second (and possibly third) Rsp5 WW domain mediates binding to the carboxyl-terminal domain (CTD) of the RNA pol II large subunit. The CTD comprises a heptamer (YSPTSPS) repeated 26 times and a PXY core that is critical for interaction with a specific group of WW domains. An analysis of synthetic peptides revealed a minimal CTD sequence that is sufficient to bind to the second Rsp5 WW domain (Rsp5 WW2) in vitro and in yeast two-hybrid assays. Furthermore, we found that specific "imperfect" CTD repeats can form a complex with Rsp5 WW2. In addition, we have shown that phosphorylation of this minimal CTD sequence on serine, threonine and tyrosine residues acts as a negative regulator of the Rsp5 WW2-CTD interaction. In view of the recent data pertaining to phosphorylation-driven interactions between the RNA pol II CTD and the WW domain of Ess1/Pin1, we suggest that CTD dephosphorylation may be a prerequisite for targeted RNA pol II degradation.
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PMID:Rsp5 WW domains interact directly with the carboxyl-terminal domain of RNA polymerase II. 1078 4

Cockayne's syndrome cells lack transcription-coupled nucleotide excision repair (TCR) and ubiquitylation of RNA polymerase II large subunit (RNA pol II LS), suggesting that ubiquitylation of RNA pol II LS may be necessary for TCR in eukaryotes. Rsp5 is the sole yeast ubiquitin-protein ligase that ubiquitylates RNA pol II LS in cells exposed to DNA-damaging agents. In yeast lacking functional Rsp5, there is no ubiquitylation of RNA pol II LS. We show here that removal, repression, or over-expression of Rsp5 has no effect on TCR, demonstrating that ubiquitylation of the RNA pol II LS is not required for TCR. We infer that the lack of ubiquitylation of RNA pol II LS in Cockayne's syndrome cells does not cause their defect in TCR.
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PMID:Transcription-coupled repair in yeast is independent from ubiquitylation of RNA pol II: implications for Cockayne's syndrome. 1090 Feb 66

The RING finger protein CNOT4 is a component of the CCR4-NOT complex. This complex is implicated in repression of RNA polymerase II transcription. Here we demonstrate that CNOT4 functions as a ubiquitin-protein ligase (E3). We show that the unique C4C4 RING domain of CNOT4 interacts with a subset of ubiquitin-conjugating enzymes (E2s). Using NMR spectroscopy, we detail the interaction of CNOT4 with UbcH5B and characterize RING residues that are critical for this interaction. CNOT4 acts as a potent E3 ligase in vitro. Mutations that destabilize the E2-E3 interface abolish this activity. Based on these results, we present a model of how E3 ligase function within the CCR4-NOT complex relates to transcriptional regulation.
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PMID:Identification of a ubiquitin-protein ligase subunit within the CCR4-NOT transcription repressor complex. 1182 28

Using an in silico (electronic database) subtraction, we identified a new member of the Ret Finger Protein-Like gene family, Rfpl4. Rfpl4 encodes a 287 amino acid putative E3 ubiquitin-protein ligase with a RING finger-like domain and a B30.2 motif. Reverse transcriptase polymerase chain reaction and Northern blot analyses reveal that Rfpl4 encodes a 1.7kb mRNA detectable exclusively in the gonads of adult mice. In situ hybridization localizes Rfpl4 transcripts within the ovary to oocytes of primary and later stage follicles and in the testis to elongating spermatids. The Rfpl4 gene comprises three exons and maps to mouse chromosome 7. We have identified the human ortholog, which maps to 19q13.4. These studies suggest that RFPL4 mediates protein degradation pathways important for gametogenesis or early embryonic development.
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PMID:The ret finger protein-like 4 gene, Rfpl4, encodes a putative E3 ubiquitin-protein ligase expressed in adult germ cells. 1185 Jan 90

The hepatitis A virus 3C protease and 3D RNA polymerase are present in low concentrations in infected cells. The 3C protease was previously shown to be rapidly degraded by the ubiquitin/26S proteasome system and we present evidence here that the 3D polymerase is also subject to ubiquitination-mediated proteolysis. Our results show that the sequence (32)LGVKDDWLLV(41) in the 3C protease serves as a protein destruction signal recognized by the ubiquitin-protein ligase E3alpha and that the destruction signal for the RNA polymerase does not require the carboxyl-terminal 137 amino acids. Both the viral 3ABCD polyprotein and the 3CD diprotein were also found to be substrates for ubiquitin-mediated proteolysis. Attempts to determine if the 3C protease or the 3D polymerase destruction signals trigger the ubiquitination and degradation of these precursors yielded evidence suggesting, but not unequivocally proving, that the recognition of the 3D polymerase by the ubiquitin system is responsible.
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PMID:Signals in hepatitis A virus P3 region proteins recognized by the ubiquitin-mediated proteolytic system. 1275 77

Monoubiquitination of histone H2B, catalyzed by Rad6-Bre1, is required for methylation of histone H3 on lysines 4 and 79, catalyzed by the Set1-containing complex COMPASS and Dot1p, respectively. The Paf1 protein complex, which associates with RNA polymerase II, is known to be required for these histone H3 methylation events. During the early elongation stage of transcription, the Paf1 complex is required for association of COMPASS with RNA polymerase II, but the role the Paf1 complex plays at the promoter has not been clear. We present evidence that the Paf1 complex is required for monoubiquitination of histone H2B at promoters. Strains deleted for several components of the Paf1 complex are defective in monoubiquitination of histone H2B, which results in the loss of methylation of lysines 4 and 79 of histone H3. We also show that Paf1 complex is required for the interaction of Rad6 and COMPASS with RNA polymerase II. Finally, we show that the Paf1 complex is required for Rad6-Bre1 catalytic activity but not for the recruitment of Rad6-Bre1 to promoters. Thus, in addition to its role during the elongation phase of transcription, the Paf1 complex appears to activate the function but not the placement of the Rad6-Bre1 ubiquitin-protein ligase at the promoters of active genes.
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PMID:The Paf1 complex is essential for histone monoubiquitination by the Rad6-Bre1 complex, which signals for histone methylation by COMPASS and Dot1p. 1287 94

The human CCR4-NOT complex is a global regulator of RNA polymerase II transcription. Recently, we showed that the RING domain CNOT4 subunit contains intrinsic ubiquitin-protein ligase (E3) activity. Here we show that binding of the CNOT4 RING finger to the ubiquitin-conjugating enzyme (E2) UbcH5B is highly selective. To understand the basis for this interaction, we identified several basic residues of UbcH5B important for binding to CNOT4 by mutational analysis. Subsequently, we tested pairs of UbcH5B and CNOT4 mutants for restoration of interaction. Concomitant charge-alteration of E49 of CNOT4 and K63 of UbcH5B restored binding and re-created a functional enzyme pair, indicative of an electrostatic interaction between these residues. The corresponding amino acids in the yeast orthologues can also be used to create a similarly designed E2-E3 enzyme pair. These are the first examples of altered-specificity E2-E3 enzyme pairs and give further insight into how E2-E3 specificity is obtained.
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PMID:An altered-specificity ubiquitin-conjugating enzyme/ubiquitin-protein ligase pair. 1500 59

The activity of the epithelial sodium (Na(+)) channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) needs to be tightly regulated to match urinary Na(+) excretion with dietary Na(+) intake. The ubiquitin-protein ligase Nedd4-2, which in vitro interacts with ENaC subunits and reduces ENaC cell surface abundance and activity by ubiquitylation of the channel, may participate in the control of ENaC. This study confirms in vivo by reverse-transcriptase-PCR that Nedd4-2 is expressed throughout the nephron and is detectable by immunoblotting in kidney extracts. By immunohistochemistry, Nedd4-2 was found to be strongly expressed in the ASDN, with low staining intensity in the late distal convoluted tubule and early connecting tubule (where apical ENaC is high) and gradually increasing detection levels toward the collecting duct (CD; where apical ENaC is low). Compared with high-Na(+) diet (5% Na(+)), 2 wk of low-Na(+) diet (0.01% Na(+)) drastically reduces Nedd4-2 immunostaining and increases apical ENaC abundance in ASDN. Reduced Nedd4-2 immunostaining is not dependent on increased apical Na(+) entry in the CD, because it is similarly observed in mice with intact and with suppressed apical ENaC activity in the CD. Consistent with a role of mineralocorticoid hormones in the long-term regulation of Nedd4-2, 5-d treatment of cultured CD (mpkCCD(cl4)) cells with 1 microM aldosterone leads to reduction of Nedd4-2 protein expression. It is concluded that Nedd4-2 abundance is regulated by Na(+) diet, by a mechanism that likely involves aldosterone. This regulation may contribute to adaptation of apical ENaC activity to altered Na(+) intake.
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PMID:Dietary sodium intake regulates the ubiquitin-protein ligase nedd4-2 in the renal collecting system. 1657 85


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