Regulation of Rho-Dependent Transcription Termination by NusG Is Specific to the <i>Escherichia coli</i> Elongation Complex

Pasman, Zvi; Hippel, Peter H. von

doi:10.1021/bi992658z

Cited by 82 publications

(85 citation statements)

References 48 publications

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“…NusG is an essential E. coli protein that regulates Rho-dependent termination by increasing the rate of transcription of elongation of E. coli RNA polymerase in the absence of Rho. This renders the transcription complex more susceptible to the termination activity of Rho and leads to early chain release (45). NusG selectively reduces transcription pausing at class II terminators, a set of pause sites where less stable RNA-DNA hybrids induce backtracking of RNA polymerase (3).…”

Section: Discussionmentioning

confidence: 99%

“…The results demonstrate that Spt5 plays a distinct role in transcription elongation by promoting the stability of transcription complexes at terminator sequences. This biochemical function is analogous to the role of the Escherichia coli RNA polymerase-binding protein NusG, a prokaryotic elongation factor that is able to stabilize prokaryotic RNA polymerase during elongation (18,45).…”

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Spt5 Cooperates with Human Immunodeficiency Virus Type 1 Tat by Preventing Premature RNA Release at Terminator Sequences

Bourgeois

Kim

Churcher

et al. 2002

Molecular and Cellular Biology

103

107

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The human immunodeficiency virus type 1 (HIV-1) Tat protein activates transcription elongation by stimulating the Tat-activated kinase (TAK/p-TEFb), a protein kinase composed of CDK9 and its cyclin partner, cyclin T1. CDK9 is able to hyperphosphorylate the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase during elongation. In addition to TAK, the transcription elongation factor Spt5 is required for the efficient activation of transcriptional elongation by Tat. To study the role of Spt5 in HIV transcription in more detail, we have developed a three-stage Tat-dependent transcription assay that permits the isolation of active preinitiation complexes, early-stage elongation complexes, and Tat-activated elongation complexes. Spt5 is recruited in the transcription complex shortly after initiation. After recruitment of Tat during elongation through the transactivation response element RNA, CDK9 is activated and induces hyperphosphorylation of Spt5 in parallel to the hyperphosphorylation of the CTD of RNA polymerase II. However, immunodepletion experiments demonstrate that Spt5 is not required for Tat-dependent activation of the kinase. Chase experiments using the Spt5-depleted extracts demonstrate that Spt5 is not required for early elongation. However, Spt5 plays an important role in late elongation by preventing the premature dissociation of RNA from the transcription complex at terminator sequences and reducing the amount of polymerase pausing at arrest sites, including bent DNA sequences. This novel biochemical function of Spt5 is analogous to the function of NusG, an elongation factor found in Escherichia coli that enhances RNA polymerase stability on templates and shows sequence similarity to Spt5.Transcription from the human immunodeficiency virus (HIV) promoter is regulated both at the level of initiation and elongation (for recent reviews, see references 14, 26, 48, and 54). In activated T cells and other permissive cell types, initiation of HIV transcription is very efficient, but production of full-length transcripts requires the viral regulatory protein Tat. In the absence of Tat, the majority of RNA polymerase II complexes that initiate transcription at the HIV promoter disengage from the template near to the promoter (24, 35). Tat strongly activates RNA polymerase processivity and permits the synthesis of full-length HIV transcripts both in vivo and in cell-free transcription systems (11,17,27,28,39,40,50,53).Tat is an RNA-binding protein that is recruited to early HIV transcription complexes by binding to the transactivation response element (TAR), an RNA stem-loop structure that is located at the 5Ј end of HIV transcripts (5, 12, 28, 51, 52). The interaction between Tat and TAR involves not only the binding of Tat to TAR RNA but also its association with the Tat-activated kinase (TAK) (20, 38, 69), a protein kinase that is able to phosphorylate the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (21,43,65).TAK is composed of a kinase subunit, CDK9, and its c...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Spt5 Cooperates with Human Immunodeficiency Virus Type 1 Tat by Preventing Premature RNA Release at Terminator Sequences

Bourgeois

Kim

Churcher

et al. 2002

Molecular and Cellular Biology

103

107

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“…For example, (i) other factors involved in transcription elongation and termination such as NusA and NusG are known to modulate Rho function (7,8,21,39,44); (ii) phage-encoded proteins Psu (from P4) and gp5.5 (from T7) are physiological antagonists of Rho and H-NS, respectively (26,27); and (iii) the cellular functions of Rho and H-NS are antagonized by overexpression of the chromosomally encoded yaeO (40) and dsrA (47) genes, respectively. There is also in vitro evidence that the activities of H-NS (43,54) and of Rho (39,57) are sensitive to the potassium salt concentration, which is known to vary within the cell with changes in osmolarity of the growth medium (10).…”

Section: Discussionmentioning

confidence: 99%

In Vivo Expression from the RpoS-Dependent P1 Promoter of the Osmotically Regulated proU Operon in Escherichia coli and Salmonella enterica Serovar Typhimurium: Activation by rho and hns Mutations and by Cold Stress

Rajkumari

Gowrishankar

2001

J Bacteriol

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Unlike the 70 -controlled P2 promoter for the osmotically regulated proU operon of Escherichia coli and Salmonella enterica serovar Typhimurium, the s -controlled P1 promoter situated further upstream appears not to contribute to expression of the proU structural genes under ordinary growth conditions. For S. enterica proU P1, there is evidence that promoter crypticity is the result of a transcription attenuation phenomenon which is relieved by the deletion of a 22-base C-rich segment in the transcript. In this study, we have sought to identify growth conditions and trans-acting mutations which activate in vivo expression from proU P1. The cryptic S. enterica proU P1 promoter was activated, individually and additively, in a rho mutant (which is defective in the transcription termination factor Rho) as well as by growth at 10°C. The E. coli proU P1 promoter was also cryptic in constructs that carried 1.2 kb of downstream proU sequence, and in these cases activation of in vivo expression was achieved either by a rho mutation during growth at 10°C or by an hns null mutation (affecting the nucleoid protein H-NS) at 30°C. The rho mutation had no effect at either 10 or 30°C on in vivo expression from two other s -controlled promoters tested, those for osmY and csiD. In cells lacking the RNA-binding regulator protein Hfq, induction of E. coli proU P1 at 10°C and by hns mutation at 30°C was still observed, although the hfq mutation was associated with a reduction in the absolute levels of P1 expression. Our results suggest that expression from proU P1 is modulated both by nucleoid structure and by Rhomediated transcription attenuation and that this promoter may be physiologically important for proU operon expression during low-temperature growth.The ProU transporter in Escherichia coli and Salmonella enterica serovar Typhimurium is a binding-protein-dependent transport system that mediates the cytoplasmic accumulation of compatible solutes such as glycine betaine, L-proline, and related compounds during growth of cells in media of elevated osmolarity (9, 10). The subunit polypeptides of the transporter are encoded by three genes, proV, proW, and proX, which together constitute (in that order) the proU operon (16).Transcription of proU in both E. coli and S. enterica is activated several-hundredfold in cultures grown in high-osmolarity media, but the mechanism of osmotic induction of the operon is not fully understood (reviewed in references 10, 19, and 29). Two cis regulatory elements that have been identified (see Fig. 1) include a 70 -driven promoter whose transcription start site is situated 60 bases upstream of proV (16, 24, 28, 54) and a negative regulatory element (NRE) approximately 500 bp long, which is situated downstream of the promoter (overlapping the proV coding region) and whose deletion results in partial derepression of proU at low osmolarity (11,24,37,38). Mutations in hns, the gene encoding an abundant nucleoid protein, H-NS, also result in partial derepression of proU expression (for a review of H-NS...

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“…1). In vitro, NusG enhances Rho termination through direct interactions with Rho and RNA polymerase (RNAP) (Li et al 1993;Pasman and von Hippel 2000;Mooney et al 2009b;Chalissery et al 2011). In vivo, NusG is proposed to either aid all Rhodependent termination (Cardinale et al 2008) or assist just a subset of terminators (Sullivan and Gottesman 1992).…”

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confidence: 99%

Rho and NusG suppress pervasive antisense transcription in Escherichia coli

et al. 2012

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Despite the prevalence of antisense transcripts in bacterial transcriptomes, little is known about how their synthesis is controlled. We report that a major function of the Escherichia coli termination factor Rho and its cofactor, NusG, is suppression of ubiquitous antisense transcription genome-wide. Rho binds C-rich unstructured nascent RNA (high C/G ratio) prior to its ATP-dependent dissociation of transcription complexes. NusG is required for efficient termination at minority subsets (~20%) of both antisense and sense Rho-dependent terminators with lower C/G ratio sequences. In contrast, a widely studied nusA deletion proposed to compromise Rho-dependent termination had no effect on antisense or sense Rho-dependent terminators in vivo. Global colocalization of the histone-like nucleoid-structuring protein (H-NS) with Rho-dependent terminators and genetic interactions between hns and rho suggest that H-NS aids Rho in suppression of antisense transcription. The combined actions of Rho, NusG, and H-NS appear to be analogous to the Sen1-Nrd1-Nab3 and nucleosome systems that suppress antisense transcription in eukaryotes.

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Regulation of Rho-Dependent Transcription Termination by NusG Is Specific to the Escherichia coli Elongation Complex

Cited by 82 publications

References 48 publications

Spt5 Cooperates with Human Immunodeficiency Virus Type 1 Tat by Preventing Premature RNA Release at Terminator Sequences

Spt5 Cooperates with Human Immunodeficiency Virus Type 1 Tat by Preventing Premature RNA Release at Terminator Sequences

In Vivo Expression from the RpoS-Dependent P1 Promoter of the Osmotically Regulated proU Operon in Escherichia coli and Salmonella enterica Serovar Typhimurium: Activation by rho and hns Mutations and by Cold Stress

Rho and NusG suppress pervasive antisense transcription in Escherichia coli

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