Identification of Functional Regions of the Nun Transcription Termination Protein of Phage HK022 and the N Antitermination Protein of Phage λ using Hybridnun-NGenes

Henthorn, Karla S.; Friedman, David I.

doi:10.1006/jmbi.1996.0142

Cited by 19 publications

(15 citation statements)

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“…The sequence conservation of Nun ARM with N ARM combined with the overall similarity of Nun-boxB and N-boxB NMR structural models suggested that their recognition strategies were indistinguishable. The activity of wtNun and casNun on and P22 reporters (Table 1) indicates boxB recognition similar to that of N (Table 3) and is consistent with previous reports of Nun (26) activities.…”

Section: Discussionsupporting

confidence: 90%

“…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 examined. N antitermination in , P22, and 21 is type specific: N proteins of one virus do not complement its absence in another (30,31), and boxBs bind noncognate N ARM peptides poorly in vitro (32)(33)(34).…”

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confidence: 99%

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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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Section: Discussionsupporting

confidence: 90%

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confidence: 99%

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

2015

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“…However, Nun induces transcription arrest, whereas N both accelerates the rate of transcription elongation and suppresses transcription termination (6). The functional differences between Nun and N are specified by their unique carboxyl-terminal regions (23). The COOH terminus of Nun is required for Nun transcription arrest both in vivo and in vitro.…”

Section: Discussionmentioning

confidence: 99%

Transcription Termination by Phage HK022 Nun Is Facilitated by COOH-terminal Lysine Residues

Kim

Gottesman

2004

Journal of Biological Chemistry

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The 109-amino acid Nun protein of prophage HK022 excludes superinfecting bacteriophage by blocking transcription elongation on the chromosome. Multiple interactions between Nun and the transcription elongation complex are involved in this reaction. The Nun NH 2 -terminal arginine-rich motif binds BOXB sequence in nascent transcripts, whereas the COOH terminus binds RNA polymerase and contacts DNA template. Nun Trp 108 is required for interaction with DNA and transcription arrest. We analyzed the role of the adjacent Lys 106 and Lys 107 residues in the Nun reaction. Substitution of the lysine residues with arginine (K106R/ K107R) had no effect on transcription arrest in vitro or in vivo. Nun K106A/K107A was partially active, whereas Nun K106D/K107D was defective in vitro and failed to exclude . All mutants bound RNA polymerase and BOXB. In contrast to Nun K106R/K107R and K106A/ K107A, Nun K106D/K107D did not cross-link DNA template. These results suggest that transcription arrest is facilitated by electrostatic interactions between positively charged Nun residues Lys 106 and Lys 107 and negatively charged DNA phosphate groups. These may assist intercalation of Trp 108 into template.

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“…We found that Nun comigrated with prestained marker proteins (Bio-Rad) in the 14-to 18-kDa range. The membranes were blocked with 5% milk powder and allowed to react overnight with antiserum against partially purified Nun raised in rabbits (antiserum kindly supplied to us by David Friedman [11]). We treated the antiserum before use with an acetone powder prepared from strain MC1000 to remove antibodies to bacterial proteins (10).…”

Section: Fig 1 Genetic Map Of the Nun-ci Region Of Hk022 (Not To Scmentioning

confidence: 99%

Constitutive Expression of a Transcription Termination Factor by a Repressed Prophage: Promoters for Transcribing the Phage HK022 nun Gene

2000

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Lysogens of phage HK022 are resistant to infection by phage . Lambda resistance is caused by the action of the HK022 Nun protein, which prematurely terminates early transcripts. We report here that transcription of the nun gene initiates at a constitutive prophage promoter, P Nun , located just upstream of the protein coding sequence. The 5 end of the transcript was determined by primer extension analysis of RNA isolated from HK022 lysogens or RNA made in vitro by transcribing a template containing the promoter with purified Escherichia coli RNA polymerase. Inactivation of P Nun by mutation greatly reduced Nun activity and Nun antigen in an HK022 lysogen. However, a low level of residual activity was detected, suggesting that a secondary promoter also contributes to nun expression. We found one possible secondary promoter, P Nun , just upstream of P Nun . Neither promoter is likely to increase the expression of other phage genes in a lysogen because their transcripts should be terminated downstream of nun. We estimate that HK022 lysogens in stationary phase contain several hundred molecules of Nun per cell and that cells in exponential phase probably contain fewer.

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Identification of Functional Regions of the Nun Transcription Termination Protein of Phage HK022 and the N Antitermination Protein of Phage λ using Hybridnun-NGenes

Cited by 19 publications

References 0 publications

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

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

Transcription Termination by Phage HK022 Nun Is Facilitated by COOH-terminal Lysine Residues

Constitutive Expression of a Transcription Termination Factor by a Repressed Prophage: Promoters for Transcribing the Phage HK022 nun Gene

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