Mechanism of sequence-specific pausing of bacterial RNA polymerase

Kireeva, Maria L.; Kashlev, Mikhail

doi:10.1073/pnas.0900407106

Cited by 108 publications

(125 citation statements)

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“…To test if Nun affects RNAP interaction with the DNA template, and to identify the site of Nun interaction with elongating RNAP, we used ExoIII, a 3′→5′ processive dsDNA exonuclease, to probe the upstream and downstream boundaries of the TECs with and without Nun. ExoIII has been widely used to determine translocation equilibria in TECs by different RNAPs and to locate DNA binding sites of proteins (32)(33)(34). Such experiments were performed as time courses, and representative time points were chosen to determine the translocation state.…”

Section: Resultsmentioning

confidence: 99%

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: Resultsmentioning

confidence: 99%

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 DNA and RNA oligonucleotides for ternary elongation complex (TEC) assembly were from IDT (Coralville, IA): RNA9, 5Ј-AUC -nitrilotriacetic acid-agarose beads, TEC purification, and elution with imidazole were performed as described (23). For the bulk elongation assay, the 5Ј-labeled RNA9 and nonlabeled TDS76 and NDS79 DNA oligonucleotides were used, and transcription assay was performed as described (25) after TEC9 purification and its elution from the beads with imidazole. To assay transcription elongation at 10 M NTP, TEC20 was obtained by the addition of 10 M each ATP, CTP, and GTP for 1 min at room temperature followed by the addition of 10 M UTP.…”

Section: Methodsmentioning

confidence: 99%

The Fidelity of Transcription

Strathern

Malagón

Irvin

et al. 2013

Journal of Biological Chemistry

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Background: Yeast RNA polymerase II domains involved in maintenance of transcription register are unknown. Results: We isolated and biochemically characterized RPB1 mutations leading to register loss (transcription slippage). Conclusion: All RPB1 mutations affect slippage localize close to the active center and near the secondary pore of RNA polymerase II. Significance: Our results shed light on the mechanism of slippage by eukaryotic RNA polymerases.

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“…Does this finding help explain the disparity in interpretation of single-molecule pausing by bacterial RNAP and yeast RNAPII (5-9)? Kireeva and Kashlev (10) show that yeast RNAPII does not recognize the C37 pause, although some other T7 DNA sites cause RNAPII to pause by backtracking. Their results clearly establish that the sequence determinants for bacterial RNAP and yeast RNAPII pausing differ.…”

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Transcriptional pausing without backtracking

Landick

2009

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

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T ranscriptional pausing by multisubunit RNA polymerases (RNAPs) plays key roles in gene regulation by coordinating RNAP movement with interactions of regulators and folding of the nascent RNA and, in metazoans, by helping program cycles of promoter-proximal transcription that poise RNAPII for gene expression (1, 2). However, mechanistic understanding of pausing is incomplete. Proposed mechanisms can be divided broadly into 2 classes ( Fig. 1): backtrack pausing, in which reverse translocation of RNAP dislodges the transcript 3Ј end from the active site and thereby prevents RNA synthesis; and (ii) nonbacktrack pausing, in which conformational rearrangements in the RNAP active site block the nucleotide addition cycle. Only a single example of nonbacktrack pausing, one for which the pause lifetime is increased by a nascent RNA hairpin, has been studied in biochemical detail (refs. 3 and 4 and references therein). Considerable disagreement exists over the contribution of nonbacktrack pauses to the ubiquitous pausing observed during both ensemble and single-molecule in vitro transcription experiments (5-9). An article in this issue of PNAS by Kireeva and Kashlev (10) now provides definitive evidence that, at least for bacterial RNAPs, significant nonbacktrack pausing occurs even without the contribution of pause RNA hairpins.Hairpin-stabilized pausing was initially studied for its role in synchronizing transcription with translation in the attenuation control regions of amino acid biosynthetic operons in enteric bacteria (e.g., the his and trp operons). Like all pauses studied to date, these sites cause kinetic partitioning of RNAPs between active and paused states (as opposed to affecting all transcribing RNAPs uniformly). These pause signals were found to be multipartite and to include synergistic contributions of different parts of RNAP's nucleic-acid scaffold both to the pause efficiency (the probability of entering the pause state at the kinetic branch between pausing and active elongation) and to pause lifetime (although the pause hairpin contributes only to pause lifetime). Detailed study of the his pause signal revealed that (i) it halted RNAP largely in the pretranslocated register ( Fig. 1; i.e., not backtracked), (ii) the active site was primarily inhibited for catalysis rather than for translocation or NTP binding, and (iii) inhibition appeared to arise by blocking formation of a 3-helix bundle composed of the RNAP bridge helix and trigger loop (4). This 3-helix bundle makes contacts to the NTP substrate in the A site required for rapid catalysis (11).A few other pause signals also have been studied to a limited extent, including (i) those in the early-transcribed region of bacteriophage T7, some of which were found to be RNA hairpinindependent (12), (ii) the Escherichia coli ops pause at which RNAP can enter backtrack states (13), and (iii) the HIV-1 ϩ62 pause (affecting human RNAPII), which appears to involve direct partitioning into a backtracked state (14). Single-molecule experiments with bacterial ...

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Mechanism of sequence-specific pausing of bacterial RNA polymerase

Cited by 108 publications

References 46 publications

Coliphage HK022 Nun protein inhibits RNA polymerase translocation

Coliphage HK022 Nun protein inhibits RNA polymerase translocation

The Fidelity of Transcription

Transcriptional pausing without backtracking

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