The Antibiotic Bicyclomycin Affects the Secondary RNA Binding Site of Escherichia coli Transcription Termination Factor Rho

Magyar, Attila; Zhang, Xiangdong; Kohn, Harold; Widger, William R.

doi:10.1074/jbc.271.41.25369

Cited by 59 publications

(131 citation statements)

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“…4). Because inhibition of secondary-site RNA binding is indicative of bicyclomycin binding to Rho (26), these results strongly indicate that bicyclomycin is unable to bind to preclosed Rho rings. Thus, bicyclomycin appears to inhibit Rho by stabilizing a conformation that is incapable of ATP hydrolysis and is incompatible with stable RNA binding to its motor regions.…”

Section: Discussionmentioning

confidence: 92%

“…S1) (24), is one such example. Previous studies have shown that bicyclomycin is a noncompetitive inhibitor of Rho ATPase activity and a mixed inhibitor of RNA binding to Rho's secondary site (25,26). Although subsequent structural studies suggested that bicyclomycin antagonizes Rho by sterically preventing the binding of the nucleophilic water molecule that initiates ATP hydrolysis (24), how bicyclomycin might interact with catalytically competent Rho states, such as those thought to accompany ATPase activity and translocation (17), has not been defined.…”

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: Discussionmentioning

confidence: 92%

mentioning

confidence: 99%

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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“…3a). Growth of strains BSNusG3 and BSNusG4 in the presence of sufficient bicyclomycin to inhibit Rho-dependent termination (Magyar et al, 1996 ;Ingham et al, 1999) did not alleviate this apparent repression of Φ(nusG-lacZ) (Fig. 3a).…”

Section: Identification Of Rif R Mutations In the Rpob Gene Of B Submentioning

confidence: 95%

Mutations in the β subunit of the Bacillus subtilis RNA polymerase that confer both rifampicin resistance and hypersensitivity to NusG

Ingham

Furneaux

2000

Microbiology

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Mutations conferring resistance to the antibiotic rifampicin (Rif r ) occur at specific sites within the β subunit of the prokaryotic RNA polymerase. Rif r mutants of Escherichia coli are frequently altered in the elongation and termination of transcription. Rif r rpoB mutations were isolated in Bacillus subtilis and their effects on transcription elongation factor NusG and Rhodependent termination were investigated. RNase protection assay, Northern analysis and the expression of nusG-lacZ fusions in cells with an inducible NusG suggested the B. subtilis nusG gene was autoregulated at the level of transcription. Rif r mutations that changed residue Q469 to a basic residue (Q469K and Q469R) enhanced autoregulation of nusG. A mutant expressing a truncated form of NusG, due to a nonsense mutation within the nusG gene, was isolated on the basis of the loss of autoregulation. The mechanism of autoregulation was found to be independent both of transcription termination factor Rho and of the promoter transcribing nusG. Autoregulation required sequences within the 5' coding sequence of the nusG gene or immediately upstream. This is the first evidence from any bacterium that Rif r RNA polymerases can display altered transcription regulation by NusG.

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“…While Rho is essential for viability in many Gram-negative organisms, Gram-positive organisms appear to be less dependent on it, although it is important in preventing transcription of noncoding RNA in the latter and has activity against Micrococcus luteus (165,166). Bicyclomycin is the only known antibiotic to target Rho, and it does not affect RNA or ATP binding to Rho but does inhibit Rho-dependent transcription termination and ATP hydrolysis (167)(168)(169). Substitutions in E. coli Rho residues L208, M219, S266, and G337 conferred resistance to bicyclomycin, suggesting that Rho was the binding target of bicyclomycin (170,171).…”

Section: Termination Factor Rhomentioning

confidence: 99%

Bacterial Transcription as a Target for Antibacterial Drug Development

Yang

Lewis

2016

Microbiol Mol Biol Rev

107

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SUMMARYTranscription, the first step of gene expression, is carried out by the enzyme RNA polymerase (RNAP) and is regulated through interaction with a series of protein transcription factors. RNAP and its associated transcription factors are highly conserved across the bacterial domain and represent excellent targets for broad-spectrum antibacterial agent discovery. Despite the numerous antibiotics on the market, there are only two series currently approved that target transcription. The determination of the three-dimensional structures of RNAP and transcription complexes at high resolution over the last 15 years has led to renewed interest in targeting this essential process for antibiotic development by utilizing rational structure-based approaches. In this review, we describe the inhibition of the bacterial transcription process with respect to structural studies of RNAP, highlight recent progress toward the discovery of novel transcription inhibitors, and suggest additional potential antibacterial targets for rational drug design.

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The Antibiotic Bicyclomycin Affects the Secondary RNA Binding Site of Escherichia coli Transcription Termination Factor Rho

Abstract: The interaction of Rho and the antibiotic bicyclomycin was probed using in vitro transcription termination reactions, poly(C) binding assays, limited tryptic digestions, and the bicyclomycin inhibition kinetics of ATPase activity in the presence of poly(dC) and ribo(C) 10

Cited by 59 publications

References 46 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

Mutations in the β subunit of the Bacillus subtilis RNA polymerase that confer both rifampicin resistance and hypersensitivity to NusG

Bacterial Transcription as a Target for Antibacterial Drug Development

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