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

Kim, Hyeong C.; Gottesman, Max E.

doi:10.1074/jbc.m313206200

Cited by 9 publications

(17 citation statements)

References 25 publications

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“…3B, compare lanes 2 and 4 and lanes 6 and 8). Conversely, when WT TEC A 11 was incubated with the nonfunctional Nun mutant (K106A/K107A) (22), arrest was inhibited to the same extent as with the mutant RNAP (Fig. 3B, compare lanes 14 and 15).…”

Section: Effect Of Nun-resistant Rnap Mutants and A Nonfunctional Nunmentioning

confidence: 99%

“…Nun is toxic upon overproduction; thus, measures were taken to ensure tight control of induction during protein expression and purification. Note that the method of Nun purification used in this study was adapted from Kim and Gottesman (22). Improvements were implemented to increase the specific activity of the full-length protein.…”

Section: Methodsmentioning

confidence: 99%

“…Improvements were implemented to increase the specific activity of the full-length protein. Nun protein was expressed from a pET-21d vector (22) and harvested from BL21 arabinose-inducible cells (Invitrogen). In these cells, expression of T7 RNAP is controlled by the stringent ara promoter.…”

Section: Methodsmentioning

confidence: 99%

“…two neighboring CTD lysines are essential for Nun activity. These residues (K106/K107) are suggested to facilitate CTD binding to template by salt-bridging and hydrogen-bonding to DNA phosphate groups (22). Although Nun has been extensively analyzed functionally, the exact mechanism by which it acts upon elongating TEC has yet to be elucidated.…”

Section: Significancementioning

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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Section: Effect Of Nun-resistant Rnap Mutants and A Nonfunctional Nunmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

See 2 more Smart Citations

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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“…First, Nun3, which blocks translation repression by the termination-defective Nun K106/107D protein, is located at nt 67 (11). Two other sequences of potential interest lie in this region of the p L transcript.…”

Section: Discussionmentioning

confidence: 99%

Role of an RNase III Binding Site in Transcription Termination at λ nutL by HK022 Nun Protein

2006

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The phage HK022 Nun protein excludes phage by binding nascent p L and p R transcripts at nutL and nutR, respectively, and inducing transcription termination just downstream of these sites. Termination is more efficient at nutL than at nutR. One difference between nutL and nutR is the presence of RNase III processing sites (rIII) located immediately promoter distal to nutL. We found that deletion of rIII dramatically reduced Nun transcription arrest in vitro but had little effect on termination in vivo. However, consistent with the in vitro results, overexpression of a transcript carrying nutL and rIII efficiently titrated Nun, allowing to grow on a strain that expressed Nun, whereas a transcript carrying only nutL or nutL-rIII with nucleotides 97 to 141 deleted was ineffective. Rnc70, an RNase III mutant that binds but does not cleave rIII, also prevented Nun-mediated exclusion. We propose that rIII enhances the on-rate of Nun at nutL, stimulating Nunmediated arrest in vitro. We have shown that a specific element in rIII, i.e., box C (G 89 GUGUGUG), strongly enhances arrest on rIII ؉ templates. Nun-rIII interactions do not stimulate Nun termination in vivo, presumably because formation of the Nun-nutL complex is normally not rate-limiting in the cell. In contrast to Nun, N is not occluded by Rnc70 and is not efficiently titrated by a nutL-rIII transcript.

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