The RING ®nger 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 C 4 C 4 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.
The NOT4 protein is a component of the CCR4⅐NOT complex, a global regulator of RNA polymerase II transcription. Human NOT4 (hNOT4) contains a RING finger motif of the C 4 C 4 type. We expressed and purified the N-terminal region of hNOT4 (residues 1-78) encompassing the RING finger motif and determined the solution structure by heteronuclear NMR. NMR experiments using a 113 Cd-substituted hNOT4 RING finger showed that two metal ions are bound through cysteine residues in a cross-brace manner. The three-dimensional structure of the hNOT4 RING finger was refined with root mean square deviation values of 0.58 ؎ 0.13 Å for the backbone atoms and 1.08 ؎ 0.12 Å for heavy atoms. The hNOT4 RING finger consists of an ␣-helix and three long loops that are stabilized by zinc coordination. The overall folding of the hNOT4 RING finger is similar to that of the C 3 HC 4 RING fingers. The relative orientation of the two zinc-chelating loops and the ␣-helix is well conserved. However, for the other regions, the secondary structural elements are distinct.The CCR4⅐NOT complex was first detected in Saccharomyces cerevisiae as a global transcription regulator, affecting transcription of multiple functionally unrelated genes positively as well as negatively (1). The complex consists of CCR4 (carbon catabolite repressor 4), CAF1 (CCR4-associated factor 1, also known as POP2), the five NOT proteins (NOT1-5), and several unidentified proteins (1). The yeast NOT genes have been identified in a screen for elevated HIS3 expression (2-4). The HIS3 gene contains two core promoters, T C , a TATA-less element, and T R , a canonical TATA sequence (5, 6). Mutations in NOT genes selectively elevate transcription from T C (2-4). Besides repressing genes involved in histidine biosynthesis (HIS3 and HIS4), NOT proteins also affect transcription of genes involved in pheromone response (STE4), nuclear fusion (BIK1), and RNA polymerase II transcription (TBP) (2, 3). The CCR4 gene product regulates expression of ADH2 and other genes involved in nonfermentative growth, cell wall integrity, and ion sensitivity (7-9). CCR4 exists in a complex with other proteins (10), and two-hybrid screening with CCR4 identified CAF1 (11, 12) and DBF2 (a cell cycle-regulated kinase) (9, 13) as binding partners. Recently, it was found that CCR4 and CAF1 reside with the NOT proteins in a 1.2-MDa complex (1). Besides physical interactions between CCR4, CAF1, and NOT proteins, there is also a functional association. Mutations in the NOT, CCR4, and CAF1 genes lead to similar, but not identical, phenotypes (1,14). Interestingly, mutations in NOT1, NOT3, NOT5, and CAF1 genes suppressed a mutation in SRB4, which is an essential component of the RNA polymerase II holoenzyme and required for the expression of most protein-coding genes. This suggests that the yeast CCR4⅐NOT complex has a very general role in RNA polymerase II transcription (15).Recently, the human counterpart of the yeast CCR4⅐NOT complex has been identified (16). cDNAs for four subunits, hNOT2, 1 hNOT3, hNOT4, and...
No abstract
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.