RNA polymerase mutants that destabilize RNA Polymerase-Promoter complexes alter NTP-sensing by rrn P1 promoters

Bartlett, Michael S.; Gaál, Tamás; Ross, Wilma; Gourse, Richard L.

doi:10.1006/jmbi.1998.1779

Cited by 101 publications

(125 citation statements)

References 53 publications

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“…Finally, sequencing of suppressors that restored prototrophy to rpoB(ΔSI1) identified two mutations in RNAP already known to suppress the amino acid requirements of ΔdksA and ppGpp 0 : rpoB(P153L) and rpoC(Δ215-220) (Fig. 1C) (7)(8)(9). The similarity of rpoB(ΔSI1) and ΔdksA across a battery of tests suggested the two mutations may have similar effects on transcription.…”

Section: Significancementioning

confidence: 98%

DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

Parshin

Shiver

Lee

et al. 2015

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

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Sensing and responding to nutritional status is a major challenge for microbial life. In Escherichia coli, the global response to amino acid starvation is orchestrated by guanosine-3′,5′-bisdiphosphate and the transcription factor DksA. DksA alters transcription by binding to RNA polymerase and allosterically modulating its activity. Using genetic analysis, photo-cross-linking, and structural modeling, we show that DksA binds and acts upon RNA polymerase through prominent features of both the nucleotide-access secondary channel and the active-site region. This work is, to our knowledge, the first demonstration of a molecular function for Sequence Insertion 1 in the β subunit of RNA polymerase and significantly advances our understanding of how DksA binds to RNA polymerase and alters transcription.transcription regulation | stringent response | protein cross-linking | molecular modeling | lineage-specific insertions S ensing and responding to nutritional status is one of the major challenges of microbial life. In Escherichia coli, the global regulatory response to amino acid starvation is orchestrated by the second messenger guanosine-3′,5′-bisdiphosphate (ppGpp), which is a widely conserved master regulator (1). Accumulation of ppGpp during amino acid scarcity triggers the stringent response, which down-regulates expression of rRNA and tRNA while increasing expression of amino acid biosynthetic enzymes. In E. coli, ppGpp works synergistically with the transcription factor DksA to initiate the stringent response (2, 3). Both ppGpp and DksA are critical for survival of stress and virulence in many pathogenic proteobacteria (4).DksA is a relatively small protein with a prominent N-terminal coiled-coil domain and a globular C-terminal domain consisting of a Zn 2+ -binding region and a C-terminal α-helix (3). It belongs to a class of regulators that bind directly to RNA polymerase (RNAP) without contacting DNA (5). DksA modulates RNAP activity by preventing formation of or destabilizing the intermediate complex (RPi) on the pathway to the open complex (RPo), which is competent for initiation. For promoters with intrinsically unstable open complexes, such as rRNA promoters, DksA binding leads to decreased transcription (2).DksA is a critical determinant of the stringent response and a model system for an important class of transcription regulators, making it essential to understand how DksA interacts with RNAP at the molecular level. High-resolution structural information of the DksA/RNAP interaction is currently unavailable. Current models agree that the coiled-coil domain of DksA inserts into the secondary channel of RNAP, the channel used by NTPs to access the active site; that the secondary channel rim helices of β' subunit are critical for DksA binding; and that residues at the tip of the coiled-coil of DksA are important for its activity. However, the precise placement of DksA is unknown. With the number of critical features that can be accessed through the secondary channel, even small changes in the model can ...

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Section: Significancementioning

confidence: 98%

DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

Parshin

Shiver

Lee

et al. 2015

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

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“…We note that solution parameters such as anion and cation concentration, temperature, template topology, etc. dramatically affect open complex half-life and, thereby, the absolute concentration of initiating NTP required for transcription (15)(16)(17). Therefore, absolute NTP concentrations required for transcription in vitro should not be extrapolated to concentrations required in vivo.…”

Section: Resultsmentioning

confidence: 99%

“…monolysogens containing promoterlacZ fusions were grown in the media described in the figure legends for 3-4 generations to an OD 600 of Ϸ0.4. Cultures were placed on ice for Ͼ30 min, lysed by sonication (16), and ␤-galactosidase activity (in Miller units) was measured (18). Where indicated, direct measurement of promoter activity was performed by primer extension.…”

Section: Methodsmentioning

confidence: 99%

“…Consistent with this observation, when purine NTP concentrations are elevated in vivo by limitation for pyrimidines, rrnB P1 promoter activity increases in parallel with the ATP concentration (15). RNAP variants and rrnB P1 promoter mutations that affect NTP requirements for transcription initiation in vitro also affect promoter regulation in vivo (16,17). Furthermore, because all seven rrn P1 promoters initiate with purine NTPs (6 with ATP and rrnD P1 with GTP), and because translation consumes both ATP and GTP, it was proposed that free purine NTP concentrations could serve as a homeostatic regulatory link between translation and ribosome synthesis (15).…”

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confidence: 99%

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NTP-sensing by rRNA promoters in Escherichia coli is direct

Schneider

Gaál

Gourse

2002

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

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105

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We showed previously that rrn P1 promoters require unusually high concentrations of the initiating nucleoside triphosphates (ATP or GTP, depending on the promoter) for maximal transcription in vitro. We proposed that this requirement for high initiating NTP concentrations contributes to control of the rrn P1 promoters from the seven Escherichia coli rRNA operons. However, the previous studies did not prove that variation in NTP concentration affects rrn P1 promoter activity directly in vivo. Here, we create conditions in vivo in which ATP and GTP concentrations are altered in opposite directions relative to one another, and we show that transcription from rrn P1 promoters that initiate with either ATP or GTP follows the concentration of the initiating NTP for that promoter. These results demonstrate that the effect of initiating NTP concentration on rrn P1 promoter activity in vivo is direct. As predicted by a model in which homeostatic control of rRNA transcription results, at least in part, from sensing of NTP concentrations by rrn P1 promoters, we show that inhibition of protein synthesis results in an increase in ATP concentration and a corresponding increase in transcription from rrnB P1. We conclude that translation is a major consumer of purine NTPs, and that NTP-sensing by rrn P1 promoters serves as a direct regulatory link between translation and ribosome synthesis.B ecause overexpression of ribosomes would be energetically costly, whereas underexpression would prevent the cell from taking full advantage of its nutritional environment, ribosome synthesis is regulated with the demand for protein synthesis. rRNA transcription is the rate-limiting step in ribosome synthesis in Escherichia coli and is controlled by several regulatory mechanisms acting at the level of transcription initiation (1, 2). In addition, an antitermination system ensures efficient rRNA transcription elongation (3).Each of the seven rRNA (rrn) operons in E. coli has two promoters, P1 and P2. The P1 promoters are responsible for the majority of rRNA transcription at moderate to fast growth rates and have been characterized extensively. Much of the intrinsic strength of the rrn P1 promoters results from AϩT-rich sequences (UP elements) upstream of the core promoters that recruit RNA polymerase (RNAP) to the promoter through specific interactions with the RNAP ␣-subunit (4-6). At least two trans-acting proteins affect rRNA transcription. Fis activates transcription from each of the 7 rrn P1 promoters by binding to sites upstream of Ϫ60 relative to the transcription start site, ϩ1 (4, 7), whereas H-NS contributes to repression of rrn P1 promoters during stationary phase (8).Although UP elements and Fis sites are required for maximal strength, rrn P1 promoters lacking these sequences (core promoters) are still regulated in response to the cell's nutritional environment (9, 10). Consistent with this finding, cells lacking the fis gene regulate transcription from rrn P1 promoters similarly to wild-type strains, because feedback systems comp...

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“…Transcription was restarted by addition of NTPs to 40 M each, and heparin to 50 g/ml. Samples were combined at 5,10,15,20,30,45,60,90,120,240, 480, and 600 s with an equal volume of stop buffer (10 M urea, 98 mM Tris borate, pH 8.3, 25 mM Na 2 EDTA, 0.05% bromphenol blue) and analyzed as described below.…”

mentioning

confidence: 99%

The Downstream DNA Jaw of Bacterial RNA Polymerase Facilitates Both Transcriptional Initiation and Pausing

Ederth¹,

Artsimovitch²,

Isaksson³

et al. 2002

Journal of Biological Chemistry

116

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Regulation of RNA polymerase during initiation, elongation, and termination of transcription is mediated in part by interactions with intrinsic regulatory signals encoded in the RNA and DNA that contact the enzyme. These interactions include contacts to an 8 -9-bp RNA: DNA hybrid within the active-site cleft of the enzyme, contacts to the melted nontemplate DNA strand in the vicinity of the hybrid, contacts to exiting RNA upstream of the hybrid, and contacts to ϳ20 bp of duplex DNA downstream of the active site. Based on characterization of an amino acid substitution (G1161R) and a deletion (⌬1149 -1190) in the jaw domain of the bacterial RNA polymerase largest subunit (␤), we report here that contacts of the jaw domain to downstream DNA at the leading edge of the transcription complex contribute to regulation during all three phases of transcription. The results provide insight into the role of the jaw domain-downstream DNA contact in transcriptional initiation and pausing and suggest possible explanations for the previously reported isolation of the jaw mutants based on reduced ColEI plasmid replication. Cellular, multisubunit RNA polymerases (RNAPs) 1 participate in a complex cycle of conformational changes to initiate, elongate, and terminate RNA transcripts. Each step in this cycle is mediated by a network of protein-nucleic acid interactions composed of interconnected parts of RNAP that contact DNA and product RNA (1-9). During elongation, most nucleic acid contacts are made by two large subunits of similar structure and sequence in prokaryotic and eukaryotic RNAPs, called ␤Ј and ␤ in bacteria or RPB1 and RPB2 in eukaryotes. During initiation, these contacts are supplemented by sequence-specific DNA contacts made by auxiliary initiation factors ( in bacteria) that mediate promoter engagement.In both initiation and elongation complexes, a key component in this protein-nucleic acid interaction network occurs between ϳ20 bp of duplex DNA downstream of the polymerization site and a channel in RNAP composed of a trough formed mostly by ␤Ј(RPB1) and a cover formed by the lobe domain of ␤(RPB2). During promoter engagement, establishment of this contact is coupled to formation of the ϳ15 bp melted transcription bubble and insertion of the template DNA strand into the active site of RNAP (Refs. 1 and 10, and references therein). Upon promoter escape, when RNAP forms a transcription elongation complex (TEC), the downstream contact persists and participates in the response of RNAP to pause, arrest, and termination signals (11-16).The downstream DNA interaction, which stretches from the position of duplex melting 1-3 nt in front of the catalytic center to ϳ20 bp further downstream, can be subdivided into activesite proximal and active-site distal sets of contacts (Fig. 1, B and C). The active-site proximal set of contacts is made at ϩ5 to ϩ8 by the lobe and domain called the clamp (formed mostly by ␤Ј), both of which can move relative to the central core of the enzyme (2, 8, 9). The active-site distal set of contacts ...

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RNA polymerase mutants that destabilize RNA Polymerase-Promoter complexes alter NTP-sensing by rrn P1 promoters

Cited by 101 publications

References 53 publications

DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

NTP-sensing by rRNA promoters in Escherichia coli is direct

The Downstream DNA Jaw of Bacterial RNA Polymerase Facilitates Both Transcriptional Initiation and Pausing

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