The Nun protein of bacteriophage HK022 inhibits translocation of <i>Escherichia coli</i>RNA polymerase without abolishing its catalytic activities

Hung, Siu Chun; Gottesman, Max E.

doi:10.1101/gad.11.20.2670

Cited by 28 publications

(34 citation statements)

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“…It has been suggested that Nun may inhibit transcription elongation by blocking RNAP translocation (8). Using a combination of bulk biochemical and presteady-state approaches, we provide evidence that translocation inhibition is, in fact, the mode of Nun action.…”

mentioning

confidence: 74%

“…Nun arrest results in the abrogation of λ transcription (8,19,20). E. coli Nus proteins facilitate Nun-mediated arrest both in vivo and in vitro.…”

Section: Discussionmentioning

confidence: 99%

“…Instead, NTP sequestration, phosphodiester bond formation, or pyrophosphate release has been suggested to be rate-limiting for transcription elongation (3,4). However, several reports strongly suggest that translocation may be at least partially rate-limiting for elongation (5)(6)(7)(8)(9).…”

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confidence: 99%

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Coliphage HK022 Nun protein inhibits RNA polymerase translocation

Vitiello

Kireeva

Lubkowska

et al. 2014

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

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The Nun protein of coliphage HK022 arrests RNA polymerase (RNAP) in vivo and in vitro at pause sites distal to phage λ N-Utilization (nut) site RNA sequences. We tested the activity of Nun on ternary elongation complexes (TECs) assembled with templates lacking the λ nut sequence. We report that Nun stabilizes both translocation states of RNAP by restricting lateral movement of TEC along the DNA register. When Nun stabilized TEC in a pretranslocated register, immediately after NMP incorporation, it prevented binding of the next NTP and stimulated pyrophosphorolysis of the nascent transcript. In contrast, stabilization of TEC by Nun in a posttranslocated register allowed NTP binding and nucleotidyl transfer but inhibited pyrophosphorolysis and the next round of forward translocation. Nun binding to and action on the TEC requires a 9-bp RNA-DNA hybrid. We observed a Nun-dependent toe print upstream to the TEC. In addition, mutations in the RNAP β′ subunit near the upstream end of the transcription bubble suppress Nun binding and arrest. These results suggest that Nun interacts with RNAP near the 5′ edge of the RNA-DNA hybrid. By stabilizing translocation states through restriction of TEC lateral mobility, Nun represents a novel class of transcription arrest factors.T ranscription elongation is highly processive, yet the rate of nucleotide addition varies significantly for different ternary elongation complexes (TECs). In the normal elongation pathway, each nucleotide addition is followed by translocation of RNA polymerase (RNAP) along DNA in single-nucleotide increments. This process transfers the RNA 3′ end from the i + 1 site to the i site of the active center, thus allowing binding of the next NTP and subsequent phosphodiester bond formation. Translocation is thought to be a stochastic, rapid, and fully reversible process and not rate-limiting for elongation (1, 2). Instead, NTP sequestration, phosphodiester bond formation, or pyrophosphate release has been suggested to be rate-limiting for transcription elongation (3, 4). However, several reports strongly suggest that translocation may be at least partially rate-limiting for elongation (5-9).Immediately following bond formation, a catalytically inactive and highly pyrophosphorolytic (pretranslocated) state is formed. In this state, the 3′ RNA end remains in the i + 1 site of the active center. The 3′ RNA thus prevents NTP binding and generates a substrate for pyrophosphorolysis. To bind the next NTP, RNAP must translocate 1-bp forward along the DNA register to form a catalytically active and pyrophosphate-resistant (posttranslocated) state. Stationary RNAP has been suggested to "ratchet" between the two states via Brownian motion (5). NTP bound to the transient posttranslocated state acts as a pawl that interferes with backward translocation, thereby favoring the formation of a phosphodiester bond. Retention of this NTP in the posttranslocated TEC is facilitated by isomerization of the active site that blocks substrate exit, and aligns it for catalysis (10, 11)...

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confidence: 74%

“…Nun arrest results in the abrogation of λ transcription (8,19,20). E. coli Nus proteins facilitate Nun-mediated arrest both in vivo and in vitro.…”

Section: Discussionmentioning

confidence: 99%

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Coliphage HK022 Nun protein inhibits RNA polymerase translocation

Vitiello

Kireeva

Lubkowska

et al. 2014

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

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“…(15). Finally, Nun arrests transcription at many sites promoterdistal to NUT (21,22). This suggests that specific amino acidnucleotide interactions are not involved in Nun transcription In general, Nun proteins mutated at residues 106 and 107 are less active at nutR than at nutL.…”

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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“…The C-terminal region makes contacts with the host NusA protein, RNA polymerase (RNAP), and DNA template (4)(5)(6). Nun induces transcription termination at sites just promoter-distal to nutL and nutR (1,7,8). Deletion of 13 C-terminal Nun amino acids stimulates NUT binding in vitro but abrogates transcription termination in vivo (6).…”

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confidence: 99%

Phage HK022 Nun protein represses translation of phage λ N (transcription termination/translation repression)

Kim¹,

Zhou²,

Wilson³

et al. 2003

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

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The N-terminal arginine-rich motif of phage HK022 Nun protein binds to NUT sequences in phage nascent transcripts and induces transcription termination. Interactions between the Nun C terminus and RNA polymerase as well as the DNA template are required for termination. We have isolated Nun C-terminal point and deletion mutants that are unable to block transcription. The mutants bind NUT RNA and inhibit translation of the N gene. Thus HK022 excludes both by terminating transcription on the phage chromosome and by preventing translation of the essential N gene. Like N autoregulation, translation repression by Nun requires host RNaseIII deficiency (rnc) or a mutation in the RNaseIII processing site (rIII) located between NUTL and the beginning of the N coding sequence. Our data support the idea that Nun bound at NUTL causes steric interference with ribosome attachment to the nearby N coding sequence. Two models, Nun acting alone or in complex with host proteins, are discussed.

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The Nun protein of bacteriophage HK022 inhibits translocation of Escherichia coliRNA polymerase without abolishing its catalytic activities

Cited by 28 publications

References 28 publications

Coliphage HK022 Nun protein inhibits RNA polymerase translocation

Coliphage HK022 Nun protein inhibits RNA polymerase translocation

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

Phage HK022 Nun protein represses translation of phage λ N (transcription termination/translation repression)

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