Ubp6 deubiquitinase controls conformational dynamics and substrate degradation of the 26S proteasome

Bashore, Charlene; Dambacher, Corey M.; Goodall, Ellen A; Matyskiela, Mary E.; Lander, Gabriel C.; Martin, Andreas

doi:10.1038/nsmb.3075

Cited by 120 publications

(203 citation statements)

References 55 publications

(94 reference statements)

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“…We also conducted the same experiments with fluorescein-labeled poly-A RNA (rA 12 *), which is a weaker secondary-site ligand than poly-U and is incompatible with primary-site binding (14,32). A clear increase for rA 12 * affinity was evident in the presence of dC 5 ( Fig. S8), especially at low ADP·BeF 3 concentrations.…”

Section: Rho Ring Closure Is Promoted By the Binding Of Pyrimidine-comentioning

confidence: 98%

“…DNA oligonucleotides [dC 5 , dC 15 , and dC 75 (dC 15 TC 15 TC 15 TC 15 TC 15 )] were purchased from IDT. FA samples were prepared, measured, and analyzed as described in SI Methods.…”

Section: Methodsmentioning

confidence: 99%

“…Although RNA binding to Rho's secondary site (the helicase translocation pore) is a major effector of ring closure, RNA binding to Rho's primary site (its OB folds) also has been implicated in controlling Rho function (27). To probe the role of primary-site ligands in modulating ring closure, we tested whether the presence or absence of dC 5 could impact the binding of rU 12 * to the secondary site. We also conducted the same experiments with fluorescein-labeled poly-A RNA (rA 12 *), which is a weaker secondary-site ligand than poly-U and is incompatible with primary-site binding (14,32).…”

Section: Rho Ring Closure Is Promoted By the Binding Of Pyrimidine-comentioning

confidence: 99%

“…5 B and C). Using fluorescein-labeled derivatives of the various primary-site ligands (denoted as dC 5 *, dC 15 *, and dC 75 *), titration experiments revealed that DNAs long enough to bridge multiple primary sites have a much higher affinity for Rho than does dC 5 (Fig. 5D); by fixing Rho at a concentration insufficient to drive ring closure with rU 12 * or rA 12 * and varying the concentration of primary-site ligands, we found that ring closure can be driven by sufficiently high concentrations of dC 5 , dC 15 , or dC 75 (Fig.…”

Section: Rho Ring Closure Is Promoted By the Binding Of Pyrimidine-comentioning

confidence: 99%

“…Many hexameric motors undergo substrate-dependent conformational changes that couple activity to the productive binding of client substrates (1)(2)(3)(4). Internal regulatory domains and exogenous proteins or small molecules frequently impact client substrate recruitment and engagement by these enzymes (5)(6)(7)(8); however, it is generally unclear how such factors control helicase or translocase dynamics.…”

mentioning

confidence: 99%

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Ligand-induced and small-molecule control of substrate loading in a hexameric helicase

Lawson

Dyer

2016

Proc. Natl. Acad. Sci. U.S.A.

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Processive, ring-shaped protein and nucleic acid protein translocases control essential biochemical processes throughout biology and are considered high-prospect therapeutic targets. The Escherichia coli Rho factor is an exemplar hexameric RNA translocase that terminates transcription in bacteria. As with many ring-shaped motor proteins, Rho activity is modulated by a variety of poorly understood mechanisms, including small-molecule therapeutics, protein-protein interactions, and the sequence of its translocation substrate. Here, we establish the mechanism of action of two Rho effectors, the antibiotic bicyclomycin and nucleic acids that bind to Rho's primary RNA recruitment site. Using small-angle X-ray scattering and a fluorescence-based assay to monitor the ability of Rho to switch between open-ring (RNA-loading) and closed-ring (RNA-translocation) states, we found bicyclomycin to be a direct antagonist of ring closure. Reciprocally, the binding of nucleic acids to its N-terminal RNA recruitment domains is shown to promote the formation of a closed-ring Rho state, with increasing primary-site occupancy providing additive stimulatory effects. This study establishes bicyclomycin as a conformational inhibitor of Rho ring dynamics, highlighting the utility of developing assays that read out protein conformation as a prospective screening tool for ring-ATPase inhibitors. Our findings further show that the RNA sequence specificity used for guiding Rho-dependent termination derives in part from an intrinsic ability of the motor to couple the recognition of pyrimidine patterns in nascent transcripts to RNA loading and activity.antibiotic | ATPase | helicase | motor protein | transcription R ing-shaped hexameric helicases and translocases are motor proteins that control myriad essential viral and cellular processes. Many hexameric motors undergo substrate-dependent conformational changes that couple activity to the productive binding of client substrates (1-4). Internal regulatory domains and exogenous proteins or small molecules frequently impact client substrate recruitment and engagement by these enzymes (5-8); however, it is generally unclear how such factors control helicase or translocase dynamics.Rho is a hexameric helicase responsible for controlling ∼20% of all transcription termination events in Escherichia coli (9). Rho is initially recruited to nascent transcripts in an open, lock washer-shaped configuration (Fig. 1A) (10, 11), where it binds preferentially to pyrimidine-rich sequences (termed "Rho utilization of termination" sequences, or "rut" sites) using a primary RNA-binding site located in the N-terminal OB folds of the hexamer (12-14). Following rut recognition, Rho converts into a closed-ring form (Fig. 1B), locking the RNA strand into a secondary RNA-binding site formed by two conserved sequence elements known as the "Q" and "R" loops (15) located within the central pore of the hexamer. This conformational change, which we show in an accompanying paper to be both RNAand ATP-dependent (16), rearran...

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Section: Rho Ring Closure Is Promoted By the Binding Of Pyrimidine-comentioning

confidence: 98%

“…DNA oligonucleotides [dC 5 , dC 15 , and dC 75 (dC 15 TC 15 TC 15 TC 15 TC 15 )] were purchased from IDT. FA samples were prepared, measured, and analyzed as described in SI Methods.…”

Section: Methodsmentioning

confidence: 99%

Section: Rho Ring Closure Is Promoted By the Binding Of Pyrimidine-comentioning

confidence: 99%

Section: Rho Ring Closure Is Promoted By the Binding Of Pyrimidine-comentioning

confidence: 99%

mentioning

confidence: 99%

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Ligand-induced and small-molecule control of substrate loading in a hexameric helicase

Lawson

Dyer

2016

Proc. Natl. Acad. Sci. U.S.A.

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TRIM24 mediates the interaction of the retinoic acid receptor alpha with the proteasome

et al. 2018

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The nuclear retinoic acid (RA) receptors (RARα, β and γ) are ligand-dependent regulators of transcription. Upon activation by RA, they are recruited at the promoters of target genes together with several coregulators. Then, they are degraded by the ubiquitin proteasome system. Here, we report that the degradation of the RARα subtype involves ubiquitination and the tripartite motif protein TRIM24, which was originally identified as a ligand-dependent corepressor of RARα. We show that in response to RA, TRIM24 serves as an adapter linking RARα to the proteasome for its degradation. In addition, TRIM24 and the proteasome are recruited with RARα to the promoters of target genes and thus are inherently linked to RARα transcriptional activity.

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Exploiting the Proteasome for Disease Treatment

Buel

Walters

2022

Protein Homeostasis in Drug Discovery

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Ubp6 deubiquitinase controls conformational dynamics and substrate degradation of the 26S proteasome

Cited by 120 publications

References 55 publications

Ligand-induced and small-molecule control of substrate loading in a hexameric helicase

Ligand-induced and small-molecule control of substrate loading in a hexameric helicase

TRIM24 mediates the interaction of the retinoic acid receptor alpha with the proteasome

Exploiting the Proteasome for Disease Treatment

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