Antisense Oligonucleotide-stimulated Transcriptional Pausing Reveals RNA Exit Channel Specificity of RNA Polymerase and Mechanistic Contributions of NusA and RfaH

Kolb, Kellie E.; Hein, Pyae P.; Landick, Robert

doi:10.1074/jbc.m113.521393

Cited by 49 publications

(79 citation statements)

References 59 publications

(89 reference statements)

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“…Because our two-hybrid data suggest that p7 and NusA do not interact directly, the stimulation of p7 binding likely results from NusA-induced conformational changes in the TEC structure. NusA may also stimulate the antipausing and antitermination activities of p7 by affecting the interaction of the β flap with the nascent RNA transcript, which has been implicated in both transcription pausing and termination (8,33).…”

Section: Discussionmentioning

confidence: 99%

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%

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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“…S4 and S5). For the hisP pause site, the RNA hairpin formation was mimicked by the addition of a complementary antisense RNA oligonucleotide (32). Transcription was started by the addition of a limited NTP set, in either the absence or presence of Gfh factors, resulting in the appearance of paused TECs and readthrough complexes stalled several nucleotides downstream of the pausing sites ( Fig.…”

Section: Gfh Factors Stimulate Transcriptional Pausing In the Presencmentioning

confidence: 99%

Regulation of transcriptional pausing through the secondary channel of RNA polymerase

Esyunina

Agapov

Kulbachinskiy

2016

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

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Transcriptional pausing has emerged as an essential mechanism of genetic regulation in both bacteria and eukaryotes, where it serves to coordinate transcription with other cellular processes and to activate or halt gene expression rapidly in response to external stimuli. Deinococcus radiodurans, a highly radioresistant and stressresistant bacterium, encodes three members of the Gre family of transcription factors: GreA and two Gre factor homologs, Gfh1 and Gfh2. Whereas GreA is a universal bacterial factor that stimulates RNA cleavage by RNA polymerase (RNAP), the functions of lineage-specific Gfh proteins remain unknown. Here, we demonstrate that these proteins, which bind within the RNAP secondary channel, strongly enhance site-specific transcriptional pausing and intrinsic termination. Uniquely, the pause-stimulatory activity of Gfh proteins depends on the nature of divalent ions (Mg 2+ or Mn 2+ ) present in the reaction and is also modulated by the nascent RNA structure and the trigger loop in the RNAP active site. Our data reveal remarkable plasticity of the RNAP active site in response to various regulatory stimuli and highlight functional diversity of transcription factors that bind inside the secondary channel of RNAP.RNA polymerase | transcriptional pausing | Gfh factors | Deinococcus radiodurans | stress response C ellular RNA polymerases (RNAPs) are complex molecular machines whose activity during transcription is regulated by DNA-and RNA-encoded signals, protein factors, small molecules, and inhibitors. The catalytic cycle of RNAP can be interrupted by pauses of various natures that play important roles in genetic regulation in all organisms, from the classic systems of transcription attenuation and their variations in bacteria (1) to recently discovered widespread promoter-proximal pausing in eukaryotes (2). The pausing serves to activate or repress transcription rapidly at specific genomic sites in response to regulatory stimuli and to coordinate RNA synthesis with other genetic processes (e.g., DNA replication and repair, RNA translation in bacteria) (1,(3)(4)(5)(6)(7)(8).Recent structural and biochemical studies revealed distinct RNAP conformations corresponding to different functional states of the transcription elongation complex (TEC) during nucleotide addition, RNA proofreading, and pausing (9-11). The control of structural transitions between these states likely underlies the function of multiple regulatory factors acting on RNAP. However, the roles of individual RNAP conformations in transcription and the mechanisms of their switching by transcription factors remain only partially understood. During transcription, RNAP holds the DNA template and the RNA transcript within its main channel, whose opening is controlled by the mobile clamp/shelf module and the flap domain of RNAP (9-11). Nucleotide addition and TEC translocation depend on alternating cycles of the folding of the trigger loop (TL) and kinking of the bridge helix (BH) in the RNAP active site (9-11) (Fig. 1A). Analysis of the stru...

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“…In particular, the hairpin-dependent pauses are enhanced by NusA via stabilization of the hairpin-RNAP interaction (12). NusA improves the termination efficiency at many hairpin-dependent terminators by facilitating the hairpin folding (13) and also by stabilizing the latter's interactions in the RNA exit channel of the transcription elongation complex (14).…”

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confidence: 99%

Transcription Elongation Factor NusA Is a General Antagonist of Rho-dependent Termination in Escherichia coli

Qayyum

Dey

Sen

2016

Journal of Biological Chemistry

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NusA is an essential protein that binds to RNA polymerase and also to the nascent RNA and influences transcription by inducing pausing and facilitating the process of transcription termination/antitermination. Its participation in Rho-dependent transcription termination has been perceived, but the molecular nature of this involvement is not known. We hypothesized that, because both Rho and NusA are RNA-binding proteins and have the potential to target the same RNA, the latter is likely to influence the global pattern of the Rho-dependent termination. Analyses of the nascent RNA binding properties and consequent effects on the Rho-dependent termination functions of specific NusA-RNA binding domain mutants revealed an existence of Rho-NusA direct competition for the overlapping nut (NusA-binding site) and rut (Rho-binding site) sites on the RNA. This leads to delayed entry of Rho at the rut site that inhibits the latter's RNA release process. High density tiling microarray profiles of these NusA mutants revealed that a significant number of genes, together with transcripts from intergenic regions, are up-regulated. Interestingly, the majority of these genes were also up-regulated when the Rho function was compromised. These results provide strong evidence for the existence of NusA-binding sites in different operons that are also the targets of Rho-dependent terminations. Our data strongly argue in favor of a direct competition between NusA and Rho for the access of specific sites on the nascent transcripts in different parts of the genome. We propose that this competition enables NusA to function as a global antagonist of the Rho function, which is unlike its role as a facilitator of hairpin-dependent termination.The bacterial transcription terminator, Rho, functions as a hexameric RNA-dependent ATPase that is capable of translocating along the RNA. It dislodges the elongating RNA polymerase (RNAP) 4 once it is loaded onto the nascent RNA (1, 2). Its termination function is facilitated upon interaction with the transcription elongation factor, NusG (3-5). Recent genomics studies have revealed that Rho-dependent termination occurs at more than one-third of the operons of a dividing Escherichia coli (6, 7). This mode of transcription termination is involved in many physiological processes, like control of translation (2), riboswitch formation (8), inhibition of unwanted antisense transcription, etc. (7). Rho is capable of loading onto unstructured stretches of RNA (the rut ([R]ho [ut]ilization) sites), and once it is bound to the RNA, it translocates in a processive manner along the 5Јto 3Ј direction, which may induce unwanted termination of transcription. Interestingly, in vivo negative regulation by cellular factors or any control switch of the Rho function has not been documented. In principle, any RNA-binding protein that has overlapping binding sites with the rut sites on the nascent RNA could influence the Rho activity by a direct competition for occupancy of the same sites.NusA, a ubiquitous transcription elo...

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Antisense Oligonucleotide-stimulated Transcriptional Pausing Reveals RNA Exit Channel Specificity of RNA Polymerase and Mechanistic Contributions of NusA and RfaH

Cited by 49 publications

References 59 publications

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

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

Regulation of transcriptional pausing through the secondary channel of RNA polymerase

Transcription Elongation Factor NusA Is a General Antagonist of Rho-dependent Termination in Escherichia coli

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