The Interaction Surface of a Bacterial Transcription Elongation Factor Required for Complex Formation with an Antiterminator during Transcription Antitermination

Mishra, Saurabh; Mohan, Shalini; Godavarthi, Sapna; Sen, Ranjan

doi:10.1074/jbc.m113.472209

Cited by 10 publications

(14 citation statements)

References 36 publications

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“…6 of Ref. 25). The presence of NusA delays the RNA loading as well as the RNA-dependent activation step(s) of Rho, which is reflected as a slow rate of RNA release by Rho.…”

Section: Nusa Binding To Nut Site(s) Delays the Loading Of Rho At Thementioning

confidence: 98%

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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“…6 of Ref. 25). The presence of NusA delays the RNA loading as well as the RNA-dependent activation step(s) of Rho, which is reflected as a slow rate of RNA release by Rho.…”

Section: Nusa Binding To Nut Site(s) Delays the Loading Of Rho At Thementioning

confidence: 98%

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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“…Jack Greenblatt first succeeded in accomplishing N action in vitro (Greenblatt, 1972), and many subsequent studies have learned much about the detailed roles of the several host proteins that participate in N-mediated antitermination (see Interaction between phage lambda and its host section below for a more detailed discussion of these factors). N protein, a natively unfolded protein (van Gilst and von Hippel, 1997; Goldenberg and Argyle, 2014), binds to the boxB sequence of the nut site in the nascent P L and P R mRNAs, as well as to the host NusA protein (Mogridge et al , 1998; Mishra et al , 2013) and RNA polymerase. It probably binds RNA polymerase on its β subunit near the RNA exit channel (Cheeran et al , 2005).…”

Section: Historical Importance and Recent Progressmentioning

confidence: 99%

“…These mutations alter several transcription factors now called NusA, NusB, NusE and NusG (originally for N under supplied but more recently N utilization substance) that are all required for successful N antitermination (Georgopoulos, 1971; Friedman, 1971; Friedman and Baron, 1974; Friedman and Gottesman, 1983; Friedman and Court, 1995). NusA is an RNA-binding protein that interacts with the α subunit of RNA polymerase and modulates transcriptional pausing (Prasch et al , 2009; Yang et al , 2009; Yang and Lewis, 2010; Schweimer et al , 2011; Zhou et al , 2011; Muteeb et al , 2012; Kolb et al , 2014), as well as interacting with lambda N protein (Mishra et al , 2013). The NusB-NusE complex binds the BoxA RNA sequence of the nut sites and binds RNA polymerase (Burmann et al , 2010; Stagno et al , 2011).…”

Section: Historical Importance and Recent Progressmentioning

confidence: 99%

Bacteriophage lambda: Early pioneer and still relevant

2015

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Molecular genetic research on bacteriophage lambda carried out during its golden age from the mid 1950's to mid 1980's was critically important in the attainment of our current understanding of the sophisticated and complex mechanisms by which the expression of genes is controlled, of DNA virus assembly and of the molecular nature of lysogeny. The development of molecular cloning techniques, ironically instigated largely by phage lambda researchers, allowed many phage workers to switch their efforts to other biological systems. Nonetheless, since that time the ongoing study of lambda and its relatives have continued to give important new insights. In this review we give some relevant early history and describe recent developments in understanding the molecular biology of lambda's life cycle.

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“…1) (4,5) and interact with host factors in the transcription elongation complex, including NusA, NusB, NusE,. N antitermination has been extensively studied, and detailed biophysical (9-11), mechanistic (12)(13)(14), and structural models are published (15)(16)(17).In competition with bacteriophage , HK022 uses its Nun protein to suppress the replication of coinfecting by premature termination of P left and P right transcripts (18-21). Similarly to N, Nun binds boxBs via its ARM in an elongation complex that also includes NusA, NusB, NusE, and NusG (6,17,20,[22][23][24][25].…”

mentioning

confidence: 99%

“…1) (4,5) and interact with host factors in the transcription elongation complex, including NusA, NusB, NusE, and NusG (6)(7)(8). N antitermination has been extensively studied, and detailed biophysical (9)(10)(11), mechanistic (12)(13)(14), and structural models are published (15)(16)(17).…”

mentioning

confidence: 99%

HK022 Nun Requires Arginine-Rich Motif Residues Distinct from λ N

2015

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Bacteriophage N protein binds boxB RNA hairpins in the nut (N utilization) sites of immediate early transcripts and interacts with host factors to suppress transcriptional termination at downstream terminators. In opposition to N, the Nun protein of HK022 binds the boxBs of coinfecting transcripts, interacts with a similar or identical set of host factors, and terminates transcription to suppress replication. Comparison of N-boxB and Nun-boxB nuclear magnetic resonance (NMR) structural models suggests similar interactions, though limited mutagenesis of Nun is available. Here, libraries of Nun's arginine-rich motif (ARM) were screened for the ability to exclude coinfection, and mutants were assayed for Nun termination with a boxB plasmid reporter system. Several Nun ARM residues appear to be immutable: Asp26, Arg28, Arg29, Arg32, Trp33, and Arg36. Asp26 and Trp33 appear to be unable to contact boxB and are not found at equivalent positions in N ARM. To understand if the requirement of Asp26, Trp33, and Arg36 indicated differences between HK022 Nun termination and N antitermination complexes, the same Nun libraries were fused to the activation domain of N and screened for clones able to complement N-deficient . Mutants were assayed for N antitermination. Surprisingly, Asp26 and Trp33 were still essential when Nun ARM was fused to N. Docking suggests that Nun ARM contacts a hydrophobic surface of the NusG carboxy-terminal domain containing residues necessary for Nun function. These findings indicate that Nun ARM relies on distinct contacts in its ternary complex and illustrate how protein-RNA recognition can evolve new regulatory functions. T he switch to delayed early gene expression in , P22, 21, and other lambdoid bacteriophages depends on N proteins assembling antitermination complexes at nut (N utilization) sites on P left and P right transcripts that transcribe past downstream terminators (1-3). N proteins bind via their arginine-rich motifs (ARMs) to boxB hairpin RNAs in nut sites of P left and P right transcripts ( Fig. 1) (4,5) and interact with host factors in the transcription elongation complex, including NusA, NusB, NusE,. N antitermination has been extensively studied, and detailed biophysical (9-11), mechanistic (12)(13)(14), and structural models are published (15)(16)(17).In competition with bacteriophage , HK022 uses its Nun protein to suppress the replication of coinfecting by premature termination of P left and P right transcripts (18-21). Similarly to N, Nun binds boxBs via its ARM in an elongation complex that also includes NusA, NusB, NusE, and NusG (6,17,20,[22][23][24][25]. Based on Nun's conserved ARM sequence, its inability to exclude P22 and 21 infections (18, 26), the affinity of Nun ARM-boxB in vitro (27,28), and the similarity of Nun's ARM-boxB NMR structural model (29) to those of N (15, 16), the recognition strategy of the HK022 Nun ARM-boxB interaction has been assumed to be very similar or identical to that of the N-boxB interaction, though few Nun ARM mutants have been ex...

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The Interaction Surface of a Bacterial Transcription Elongation Factor Required for Complex Formation with an Antiterminator during Transcription Antitermination

Cited by 10 publications

References 36 publications

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

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

Bacteriophage lambda: Early pioneer and still relevant

HK022 Nun Requires Arginine-Rich Motif Residues Distinct from λ N

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