Role of the Coiled-Coil Tip of Escherichia coli DksA in Promoter Control

Lee, Jeong‐Hyun; Lennon, Christopher; Ross, Wilma; Gourse, Richard L.

doi:10.1016/j.jmb.2011.12.028

Cited by 42 publications

(79 citation statements)

References 36 publications

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“…This assignment is supported by cross-linking between DksA-V73 and the overlapping region (β653-681) as well as functional analysis of mutants in D74, β-R678, and β-R1106. We found that DksA-D74 substitutions D74N, D74S, and D74E all altered DksA activity without affecting binding, consistent with previous studies (3,12,21) (Fig. 5B and SI Appendix, Fig.…”

Section: Significancesupporting

confidence: 91%

“…Our experimental efforts focused on the effects of DksA during initiation, but the position of the DksA coiled-coil in the channel may be dynamic and vary with RNAP conformation, the stage of transcription cycle, and the presence of additional factors such as ppGpp. Indeed, Fe 2+ -mediated cleavage of DksA is reduced for nonfunctional DksA-tip mutants (21), and in the paused complex as compared to free RNAP (20). This reduced cleavage has been interpreted as representing a more distal position of the coiled-coil in the these complexes (20).…”

Section: Discussionmentioning

confidence: 99%

“…We note that the proposed interaction of DksA-D74 with the substrate-binding region of the active site places DksA-A76 tightly against β'G782/L783 from the bridge helix. This provides an alternative explanation for the functional defects discovered for DksA-A76T, in that a steric clash from the bulky substitution would prevent DksA from inserting into the substrate-binding region of the active site (21).…”

Section: Significancementioning

confidence: 98%

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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: Significancesupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Section: Significancementioning

confidence: 98%

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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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“…The active residue of DksA (aspartic acid 74) is located at the tip of the coiled-coil domain, which would contact the trigger loop (TL) of RNAP. Such an interaction was recently tested using various DksA-GreA/B chimeras where the Gre factors could mimic DksA and initiate transcription when equipped with a DksA-like active tip (37). Additionally, DksA acts antagonistically to GreA in the regulation of rRNA promoters, which suggests that both molecules compete for the same surface on RNAP (38)(39)(40)(41).…”

mentioning

confidence: 99%

Characterization of a Novel RNA Polymerase Mutant That Alters DksA Activity

Satory

Halliday²,

Sivaramakrishnan³

et al. 2013

Journal of Bacteriology

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The auxiliary factor DksA is a global transcription regulator and, with the help of ppGpp, controls the nutritional stress response in Escherichia coli. Although the consequences of its modulation of RNA polymerase (RNAP) are becoming better explained, it is still not fully understood how the two proteins interact. We employed a series of genetic suppressor selections to find residues in RNAP that alter its sensitivity to DksA. Our approach allowed us to identify and genetically characterize in vivo three single amino acid substitutions: ␤= E677G, ␤ V146F, and ␤ G534D. We demonstrate that the mutation ␤= E677G affects the activity of both DksA and its homolog, TraR, but does not affect the action of other secondary interactors, such as GreA or GreB. Our mutants provide insight into how different auxiliary transcription factors interact with RNAP and contribute to our understanding of how different stages of transcription are regulated through the secondary channel of RNAP in vivo. The vast majority of known transcription factors modulate gene activity by mediating the binding of RNA polymerase (RNAP) to promoters and other regulatory DNA sequences. Another class, the auxiliary transcription factors, interacts directly with the RNAP complex instead of binding DNA. Bacterial auxiliary transcription factors that bind to the secondary channel of RNAP share a set of common features: they are small proteins (up to about 150 amino acids) that consist of a globular head and a coiled-coil domain (1). Low-resolution cryo-electron microscopy data have revealed that the globular domain interacts with the surface of RNAP and the coiled-coil protrudes deep into the secondary channel of the RNAP complex (2-4). The secondary channel is an opening formed by the ␤ and ␤= subunits, through which free ribonucleoside triphosphates access the catalytic center of the enzyme. The common structure of this class of transcription factors allows them to reach the active site through the secondary channel and modulate the RNAP. Several such secondary channel interactors have been identified to date in bacteria: transcription elongation and fidelity factors GreA and GreB (5-9); two anti-Gre factors, Gfh1 (10, 11) and Rnk (12); and the transcription initiation and elongation factor DksA (13-16) and its analog, DksA2, from Pseudomonas aeruginosa (17), as well as its structural homolog, TraR, often found on conjugative plasmids (18).A considerable amount of work has been done to comprehend the role of DksA in Escherichia coli. Recent evidence has prompted a shift in the understanding of its function: its role has evolved from a transcriptional cofactor to a major player regulating transcription at multiple levels. DksA was originally isolated as a suppressor of the mutant phenotype resulting from the loss of function of the chaperone DnaK (19). The involvement of DnaK in restarting stalled DNA polymerase suggests a probable role for DksA in replication (20). In fact, DksA was recently shown to play a role in prevention of the collision between ...

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“…DksA exerts its effects by binding directly to the secondary channel of RNAP and allosterically modifying the kinetic properties of the enzyme, thereby sensitizing RNAP to changes in the concentrations of guanosine tetraphosphate (ppGpp) and the initiating nucleoside triphosphate (iNTP) (26,29,34,35). D74, an aspartate residue at the tip of the coiled-coil domain, is necessary for DksA to function in transcription initiation (16). In addition, DksA aids in resolving conflicts between the active DNA replication machinery and stalled or elongating RNA polymerases, and it helps to ensure the proper resolution of double-strand breaks caused by these collisions (22,42,45).…”

mentioning

confidence: 99%

Suppression of a dnaKJ Deletion by Multicopy dksA Results from Non-Feedback-Regulated Transcripts That Originate Upstream of the Major dksA Promoter

et al. 2012

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cDksA is an RNA polymerase (RNAP) binding transcription factor that controls expression of a large number of genes in concert with the small-molecule "alarmone" ppGpp. DksA also aids in the resolution of conflicts between RNAP and DNA polymerase (DNAP) during genome replication. DksA was originally identified as a multicopy suppressor of the temperature sensitivity caused by deletion of the genes coding for the DnaKJ chaperone system. Here, we address a longstanding question regarding the role of DksA in ⌬dnaKJ suppression. We demonstrate that DksA expression from a multicopy plasmid is necessary and sufficient for suppression, that overexpression occurs despite the fact that the major dksA promoter is feedback regulated in wildtype cells, and that weak, non-feedback-regulated transcription originating upstream of the major promoter for the dksA gene accounts for overexpression. We tentatively rule out three potential explanations for suppression related to known functions of DnaKJ. Because a determinant in DksA needed for the regulation of transcription initiation, but not for resolution of RNAP-DNAP conflicts, is needed to bypass the need for DnaKJ, we suggest that suppression results from an unidentified product whose promoter is directly or indirectly regulated by DksA.

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Role of the Coiled-Coil Tip of Escherichia coli DksA in Promoter Control

Cited by 42 publications

References 36 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

Characterization of a Novel RNA Polymerase Mutant That Alters DksA Activity

Suppression of a dnaKJ Deletion by Multicopy dksA Results from Non-Feedback-Regulated Transcripts That Originate Upstream of the Major dksA Promoter

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