A Conserved Zinc Binding Domain in the Largest Subunit of DNA-dependent RNA Polymerase Modulates Intrinsic Transcription Termination and Antitermination but does not Stabilize the Elongation Complex

King, Rodney A.; Markov, Dmitry; Sen, Ranjan; Severinov, Konstantin; Weisberg, Robert A.

doi:10.1016/j.jmb.2004.07.072

Cited by 33 publications

(23 citation statements)

References 34 publications

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“…S8). This corresponds to the previously reported effects of β′ ZBD mutations on intrinsic termination by E. coli RNAP (20). At the same time, p7 suppressed termination by the mutant RNAP to almost the same level as in the case of the control reconstituted wild-type X. oryzae RNAP.…”

supporting

confidence: 90%

“…1A and Table S2): (i) the β′ coiled-coil/β gate loop, targeted by RfaH and its paralogs (15); (ii) the β flap/RNA exit channel, targeted by N, Q and gp39 (4, 7, 9-11); and (iii) the β′ N terminus and adjacent regions, involved in antitermination by p7. In addition, p7 seems to partially share its target site with an RNA-based antiterminator, the put hairpin encoded by phage HK022, the function of which was shown to depend on the β′ ZBD (20,43,44). Despite the different binding sites, all classes of factors suppress transcription pausing by bacterial RNAP, which therefore is likely a universal part of all antitermination mechanisms (Table S2).…”

Section: Discussionmentioning

confidence: 99%

“…1A), suggesting that the mechanism of transcription antitermination by p7 may differ from those used by other phage antiterminator proteins (17,19). Previously, the β′ ZBD, which together with the β flap forms the RNA exit channel, has been implicated in transcription termination and antitermination (20), whereas no termination-specific functions have been reported for the ω subunit. A recent study suggested that p7 can also interact with the β flap domain, which may be important for inhibition of transcription initiation (17).…”

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confidence: 99%

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Distinct pathways of RNA polymerase regulation by a phage-encoded factor

Esyunina

Klimuk

Severinov

et al. 2015

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

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Transcription antitermination is a common strategy of gene expression regulation, but only a few transcription antitermination factors have been studied in detail. Here, we dissect the transcription antitermination mechanism of Xanthomonas oryzae virus Xp10 protein p7, which binds host RNA polymerase (RNAP) and regulates both transcription initiation and termination. We show that p7 suppresses intrinsic termination by decreasing RNAP pausing and increasing the transcription complex stability, in cooperation with host-encoded factor NusA. Uniquely, the antitermination activity of p7 depends on the ω subunit of the RNAP core and is modulated by ppGpp. In contrast, the inhibition of transcription initiation by p7 does not require ω but depends on other RNAP sites. Our results suggest that p7, a bifunctional transcription factor, uses distinct mechanisms to control different steps of transcription. We propose that regulatory functions of the ω subunit revealed by our analysis may extend to its homologs in eukaryotic RNAPs.RNA polymerase | transcription antitermination | transcription pausing | omega subunit | NusA T ranscription promoters and terminators are the main "punctuation marks" that control the expression of individual genes in the genome. In bacteria, transcription termination is an essential regulatory step that determines the relative levels of expression of promoter-proximal and distal genes within operons. Depending on the factors involved, transcription termination in bacteria can be classified as intrinsic (depends on RNAencoded signals) or factor-dependent (requires additional protein factors such as Rho helicase). Intrinsic terminators consist of a G/C-rich hairpin formed in the nascent RNA followed by a downstream oligo(U) tract. During intrinsic termination, RNA polymerase (RNAP) pauses after synthesizing an oligo(U) sequence, accompanied by hairpin formation, leading to subsequent dissociation of the transcription elongation complex (TEC) (1, 2). Despite the relatively simple overall scenario, the exact mechanisms of pausing and hairpin-induced TEC destabilization remain an issue of debate (1, 3).Transcription antitermination is a widespread phenomenon involved in regulation of bacterial and phage operons (2). Transcription antitermination is often mediated by specialized protein factors that target host RNAP and can suppress termination at multiple sites in the operon. The well-studied examples of phage antiterminators include proteins N and Q of Escherichia coli phage λ and the gp39 protein of Thermus thermophilus phage P23-45. These factors suppress RNAP pausing, thus inhibiting the first step of the termination pathway (4-6). The antitermination activity of N and Q, but not gp39, is enhanced by cell-encoded proteins, including transcription elongation factor NusA, which by itself stimulates termination but reverses its activity to additionally stabilize the TEC in cooperation with N and Q (5, 6). Curiously, the N, Q, gp39, and NusA proteins all target the flexible β flap domain of RNAP (2, 4...

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Section: Discussionmentioning

confidence: 99%

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Distinct pathways of RNA polymerase regulation by a phage-encoded factor

Esyunina

Klimuk

Severinov

et al. 2015

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

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“…One arm of each put RNA binds directly to RNAP throughout elongation; put -modified enzymes transcribe at faster rates than enzymes that are not bound by put RNAs and read through terminators located thousands of base pairs downstream 86 . The activity of the put transcripts can be reconstituted in vitro in the absence of accessory factors and is thought to be mediated by a direct interaction with the β′-zinc-finger 87 . The put RNAs inhibit RNAP backtracking near the site of recruitment by blocking the re-entry of the transcript into the RNA exit channel 88 , but the mechanism of long-range antitermination modification and the involvement of any cellular proteins remain unknown.…”

Section: Bypassing Multiple Consecutive Terminatorsmentioning

confidence: 99%

Termination and antitermination: RNA polymerase runs a stop sign

2011

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Termination signals induce rapid and irreversible dissociation of the nascent transcript from RNA polymerase. Terminators at the end of genes prevent unintended transcription into the downstream genes, whereas terminators in the upstream regulatory leader regions adjust expression of the structural genes in response to metabolic and environmental signals. Premature termination within an operon leads to potentially deleterious defects in the expression of the downstream genes, but also provides an important surveillance mechanism. This Review discusses the actions of bacterial and phage antiterminators that allow RNA polymerase to override a terminator when the circumstances demand it.

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“…The Zn-binding element is in close proximity to the RNA exit channel; p7 may thus influence transcription termination by altering the Zn-binding element interactions with RNA. In this scenario, the p7 mechanism of action would be similar to transcription antitermination by the phage HK022 put element, which acts by affecting the Zn-binding element interactions with the transcript (17). The β′-zipper is located at the upstream edge of transcription bubble, where the non-template DNA strand controls transcription termination efficiency by displacing the RNA from the RNA-DNA hybrid (18).…”

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Mapping of RNA Polymerase Residues that Interact with Bacteriophage Xp10 Transcription Antitermination Factor p7

Yuzenkova

Zenkin

Severinov

2008

Journal of Molecular Biology

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Bacteriophage Xp10-encoded transcription factor p7 interacts with host Xanthomonas oryzae RNA polymerase β′ subunit and prevents both promoter recognition by the RNA polymerase holoenzyme and transcription termination by the RNA polymerase core. P7 does not bind to and has no effect on RNA polymerase from E. coli. Here, we use a combination of biochemical and genetic methods to map the p7 interaction site to within four β′ amino acids at the N-terminus of X. oryzae RNAP β′. The interaction site is located in an area that is close to the promoter spacer in the open complex and to the upstream boundary of the transcription bubble in the elongation complex, providing possible explanation as to how a small protein can affect both transcription initiation and termination by binding to the same RNA polymerase site. KeywordsRNA polymerase; transcription antitermination; promoter recognition; transcription factor; bacteriophage In bacteria, the regulation of activity of DNA-dependent RNA polymerase (RNAP), the key enzyme of transcription, is often accomplished through interactions with protein transcription factors. The RNAP core enzyme (subunit composition α 2 β β′ω) is catalytically proficient (i.e., it can perform transcription elongation and terminate transcription on intrinsic terminators) but cannot initiate transcription from promoters. When a σ subunit binds the core, an RNAP holoenzyme capable of specific initiation from promoters is formed.Most bacteriophages rely on RNAP of their host bacteria for expression of their genes during the infection process. Coordinate expression of various classes of viral genes is accomplished through the action of bacteriophage-encoded transcription factors that bind to and regulate the properties of host RNAP. Bacteriophage Xp10 (1,2) infects Xanthomonas oryzae, a rice *Corresponding author, Waksman Institute for Microbiology, 190 Frelinghuysen Road, Piscataway, NJ, 08854, Phone: (732) FAX: (732) 445-5735, E-mail: severik@waksman.rutgers.edu. $ Present address: Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript pathogen (3). A 73-amino-acid-long Xp10 protein p7 binds to and inhibits X. oryzae RNAP (4) and therefore may be responsible for the shut-off of host transcription that is known to occur during Xp10 infection (5). P7 is a novel protein, with no similarity to proteins of known functions in public databases. P7 in...

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A Conserved Zinc Binding Domain in the Largest Subunit of DNA-dependent RNA Polymerase Modulates Intrinsic Transcription Termination and Antitermination but does not Stabilize the Elongation Complex

Cited by 33 publications

References 34 publications

Distinct pathways of RNA polymerase regulation by a phage-encoded factor

Distinct pathways of RNA polymerase regulation by a phage-encoded factor

Termination and antitermination: RNA polymerase runs a stop sign

Mapping of RNA Polymerase Residues that Interact with Bacteriophage Xp10 Transcription Antitermination Factor p7

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