Coliphage HK022 Nun protein inhibits RNA polymerase translocation

Vitiello, Carmen; Kireeva, Maria L.; Lubkowska, Lucyna; Kashlev, Mikhail; Gottesman, Max E.

doi:10.1073/pnas.1319740111

Cited by 29 publications

(57 citation statements)

References 45 publications

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“…During backtracking, the RNA 3′ terminus moves from the catalytic center into the secondary channel, thus rendering TEC catalytically inactive. Bacterial transcription factors (TFs) GreA and GreB (6-10), as well as the analogous eukaryotic TFIIS factor (11, 12), rescue backtracked TEC by promoting degradation of this disengaged RNA, thus restoring the 3′ end of the transcript within the RNAP active center and allowing transcription to proceed.The 12-kDa Nun protein of coliphage HK022 excludes phage λ superinfection by specifically blocking TEC translocation on λ DNA (13,14). Nun competes with the λ N protein, which, conversely, enhances elongation and suppresses transcription termination.…”

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confidence: 99%

“…Furthermore, mutations in the RNAP β′ subunit near the upstream end of the transcription bubble (Fig. 1C) suppress Nun binding and arrest (14,17).Despite the numerous biochemical studies of the effect of Nun on transcription, little is known about the structural mechanism of Nun action. In the present study we used a cross-linking approach to probe Nun interactions with the RNA transcript and TEC.…”

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confidence: 99%

“…The 12-kDa Nun protein of coliphage HK022 excludes phage λ superinfection by specifically blocking TEC translocation on λ DNA (13,14). Nun competes with the λ N protein, which, conversely, enhances elongation and suppresses transcription termination.…”

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confidence: 99%

“…The carboxylterminal domain (CTD) of Nun or λ N interacts with RNAP to block or stimulate transcription elongation, respectively (13). At high Nun concentrations, however, transcription arrest occurs in vitro or in vivo on templates lacking the λ NUT sequence (14,15). Interaction between the Nun CTD and RNAP was probed using several different approaches.…”

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confidence: 99%

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Probing the structure of Nun transcription arrest factor bound to RNA polymerase

Mustaev¹,

Vitiello²,

Gottesman³

2016

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

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The coliphage HK022 protein Nun transcription elongation arrest factor inhibits RNA polymerase translocation. In vivo, Nun acts specifically to block transcription of the coliphage λ chromosome. Using in vitro assays, we demonstrate that Nun cross-links RNA in an RNA:DNA hybrid within a ternary elongation complex (TEC). Both the 5′ and the 3′ ends of the RNA cross-link Nun, implying that Nun contacts RNA polymerase both at the upstream edge of the RNA:DNA hybrid and in the vicinity of the catalytic center. This finding suggests that Nun may inhibit translocation by more than one mechanism. Transcription elongation factor GreA efficiently blocked Nun cross-linking to the 3′ end of the transcript, whereas the highly homologous GreB factor did not. Surprisingly, both factors strongly suppressed Nun cross-linking to the 5′ end of the RNA, suggesting that GreA and GreB can enter the RNA exit channel as well as the secondary channel, where they are known to bind. These findings extend the known action mechanism for these ubiquitous cellular factors.Nun protein | Gre factors | transcription arrest | phage HK022 | ternary elongation complex T ranscription elongation by RNA polymerase (RNAP) is regulated by pausing and termination signals encoded in the transcribed DNA sequence, as well as by numerous protein factors and free RNA molecules. In transcribing ternary elongation complexes (TEC) (Fig. 1A), the RNA product remains transiently bound to a template DNA strand, forming a RNA:DNA hybrid 9-to 10-bp long (1). Transcription, although processive, is frequently interrupted by RNAP pausing. Mechanisms of such pausing include intrinsic pausing (2, 3) and backtracking (1, 4, 5), the process by which RNAP reverse-translocates. During backtracking, the RNA 3′ terminus moves from the catalytic center into the secondary channel, thus rendering TEC catalytically inactive. Bacterial transcription factors (TFs) GreA and GreB (6-10), as well as the analogous eukaryotic TFIIS factor (11, 12), rescue backtracked TEC by promoting degradation of this disengaged RNA, thus restoring the 3′ end of the transcript within the RNAP active center and allowing transcription to proceed.The 12-kDa Nun protein of coliphage HK022 excludes phage λ superinfection by specifically blocking TEC translocation on λ DNA (13,14). Nun competes with the λ N protein, which, conversely, enhances elongation and suppresses transcription termination. The amino-terminal RNA-binding domain (arginine-rich motif, or ARM) (Fig. 1B) is similar and plays the same role for both proteins: that of binding to the λ nascent transcript NUT sequence. The carboxylterminal domain (CTD) of Nun or λ N interacts with RNAP to block or stimulate transcription elongation, respectively (13). At high Nun concentrations, however, transcription arrest occurs in vitro or in vivo on templates lacking the λ NUT sequence (14, 15). Interaction between the Nun CTD and RNAP was probed using several different approaches. The CTD conjugated to a photoreactive group cross-links with a DNA template ...

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Probing the structure of Nun transcription arrest factor bound to RNA polymerase

Mustaev¹,

Vitiello²,

Gottesman³

2016

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

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“…In competition with bacteriophage , HK022 uses its Nun protein to suppress the replication of coinfecting by premature termination of P left and P right transcripts (18)(19)(20)(21). Similarly to N, Nun binds boxBs via its ARM in an elongation complex that also includes NusA, NusB, NusE, and NusG (6,17,20,(22)(23)(24)(25).…”

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HK022 Nun Requires Arginine-Rich Motif Residues Distinct from λ N

2015

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Bacteriophage N protein binds boxB RNA hairpins in the nut (N utilization) sites of immediate early transcripts and interacts with host factors to suppress transcriptional termination at downstream terminators. In opposition to N, the Nun protein of HK022 binds the boxBs of coinfecting transcripts, interacts with a similar or identical set of host factors, and terminates transcription to suppress replication. Comparison of N-boxB and Nun-boxB nuclear magnetic resonance (NMR) structural models suggests similar interactions, though limited mutagenesis of Nun is available. Here, libraries of Nun's arginine-rich motif (ARM) were screened for the ability to exclude coinfection, and mutants were assayed for Nun termination with a boxB plasmid reporter system. Several Nun ARM residues appear to be immutable: Asp26, Arg28, Arg29, Arg32, Trp33, and Arg36. Asp26 and Trp33 appear to be unable to contact boxB and are not found at equivalent positions in N ARM. To understand if the requirement of Asp26, Trp33, and Arg36 indicated differences between HK022 Nun termination and N antitermination complexes, the same Nun libraries were fused to the activation domain of N and screened for clones able to complement N-deficient . Mutants were assayed for N antitermination. Surprisingly, Asp26 and Trp33 were still essential when Nun ARM was fused to N. Docking suggests that Nun ARM contacts a hydrophobic surface of the NusG carboxy-terminal domain containing residues necessary for Nun function. These findings indicate that Nun ARM relies on distinct contacts in its ternary complex and illustrate how protein-RNA recognition can evolve new regulatory functions. T he switch to delayed early gene expression in , P22, 21, and other lambdoid bacteriophages depends on N proteins assembling antitermination complexes at nut (N utilization) sites on P left and P right transcripts that transcribe past downstream terminators (1-3). N proteins bind via their arginine-rich motifs (ARMs) to boxB hairpin RNAs in nut sites of P left and P right transcripts ( Fig. 1) (4,5) and interact with host factors in the transcription elongation complex, including NusA, NusB, NusE,. N antitermination has been extensively studied, and detailed biophysical (9-11), mechanistic (12)(13)(14), and structural models are published (15)(16)(17).In competition with bacteriophage , HK022 uses its Nun protein to suppress the replication of coinfecting by premature termination of P left and P right transcripts (18-21). Similarly to N, Nun binds boxBs via its ARM in an elongation complex that also includes NusA, NusB, NusE, and NusG (6,17,20,[22][23][24][25]. Based on Nun's conserved ARM sequence, its inability to exclude P22 and 21 infections (18, 26), the affinity of Nun ARM-boxB in vitro (27,28), and the similarity of Nun's ARM-boxB NMR structural model (29) to those of N (15, 16), the recognition strategy of the HK022 Nun ARM-boxB interaction has been assumed to be very similar or identical to that of the N-boxB interaction, though few Nun ARM mutants have been ex...

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2020

Structure and Function of the Bacterial Genome

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Coliphage HK022 Nun protein inhibits RNA polymerase translocation

Cited by 29 publications

References 45 publications

Probing the structure of Nun transcription arrest factor bound to RNA polymerase

Probing the structure of Nun transcription arrest factor bound to RNA polymerase

HK022 Nun Requires Arginine-Rich Motif Residues Distinct from λ N

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