DksA2, a zinc‐independent structural analog of the transcription factor DksA

Furman, Ran; Biswas, Tapan; Danhart, Eric; Foster, Mark P.; Tsodikov, Oleg V.; Artsimovitch, Irina

doi:10.1016/j.febslet.2013.01.073

Cited by 35 publications

(51 citation statements)

References 29 publications

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“…This and previous studies have shown via complementation or transcription experiments that when DksA has a DXXDXA motif at the tip of the CC domain, it functions as a transcriptional regulator (5,15,17,18). Accordingly, DksA activity is lost when the second D in the motif is mutated (14,(17)(18)(19).…”

Section: Discussionsupporting

confidence: 68%

“…Previous work showed that the aspartates as well as the alanine in the DXXDXA motif are crucial for activity (14,15,17,18). We constructed variants of DksA and SMc00049, modifying the motif, to investigate if we could abolish or establish DksA activity.…”

Section: Resultsmentioning

confidence: 99%

“…The DksA globular domain has been shown to bind to the rim helices located in the ␤= subunit of RNAP (15). It is formed by the C and N termini of DksA and harbors a CXXCX 17 CXXC zinc finger motif (13). In addition, recent work in E. coli identified the RNAP ␤ subunit sequence in-sertion 1 as a binding site for the DksA C-terminal helix (16).…”

mentioning

confidence: 99%

“…In addition, recent work in E. coli identified the RNAP ␤ subunit sequence in-sertion 1 as a binding site for the DksA C-terminal helix (16). Structural as well as amino acid substitution analyses of DksA proteins from Pseudomonas aeruginosa, Rhodobacter sphaeroides, and E. coli indicate that the conserved amino acid motif DXXDXA at the tip of the CC domain is critical for DksA function as a transcriptional regulator (17)(18)(19). The globular domain with conserved cysteines seems to play an important role in P. aeruginosa DksA2 (17) and might be involved in proper folding of E. coli DksA (18).…”

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confidence: 99%

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Contributions of Sinorhizobium meliloti Transcriptional Regulator DksA to Bacterial Growth and Efficient Symbiosis with Medicago sativa

Wippel

Long

2016

J Bacteriol

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The stringent response, mediated by the (p)ppGpp synthetase RelA and the RNA polymerase-binding protein DksA, is triggered by limiting nutrient conditions. For some bacteria, it is involved in regulation of virulence. We investigated the role of two DksA-like proteins from the Gram-negative nitrogen-fixing symbiont Sinorhizobium meliloti in free-living culture and in interaction with its host plant Medicago sativa. The two paralogs, encoded by the genes SMc00469 and SMc00049, differ in the constitution of two major domains required for function in canonical DksA: the DXXDXA motif at the tip of a coiled-coil domain and a zinc finger domain. Using mutant analyses of single, double, and triple deletions for SMc00469 (designated dksA), SMc00049, and relA, we found that the ⌬dksA mutant but not the ⌬SMc00049 mutant showed impaired growth on minimal medium, reduced nodulation on the host plant, and lower nitrogen fixation activity in early nodules, while its nod gene expression was normal. The ⌬relA mutant showed severe pleiotropic phenotypes under all conditions tested. Only S. meliloti dksA complemented the metabolic defects of an Escherichia coli dksA mutant. Modifications of the DXXDXA motif in SMc00049 failed to establish DksA function. Our results imply a role for transcriptional regulator DksA in the S. meliloti-M. sativa symbiosis. IMPORTANCEThe stringent response is a bacterial transcription regulation process triggered upon nutritional stress. Sinorhizobium meliloti, a soil bacterium establishing agriculturally important root nodule symbioses with legume plants, undergoes constant molecular adjustment during host interaction. Analyzing the components of the stringent response in this alphaproteobacterium helps understand molecular control regarding the development of plant interaction. Using mutant analyses, we describe how the lack of DksA influences symbiosis with Medicago sativa and show that a second paralogous S. meliloti protein cannot substitute for this missing function. This work contributes to the field by showing the similarities and differences of S. meliloti DksA-like proteins to orthologs from other species, adding information to the diversity of the stringent response regulatory system. Bacteria employ the stringent response as a mechanism to adjust global gene expression to adverse nutrient conditions. For example, when Escherichia coli encounters amino acid starvation, the ribosome-bound protein RelA (1, 2) recognizes uncharged tRNA molecules and synthesizes guanosine tetraphosphate and guanosine pentaphosphate (referred to here as ppGpp) from GTP and ATP (3). The alarmone ppGpp and the regulatory protein DksA subsequently bind RNA polymerase (RNAP) and alter the kinetic properties of promoter/RNAP complexes (4). In particular, ppGpp and DksA reduce the lifetime of promoter/RNAP complexes by inhibiting the transition from closed to intermediate complex formation on promoters depending on the primary sigma factor RpoD ( 70 ) (4, 5). In addition, RNAP dissociation can allow alternative...

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Section: Discussionsupporting

confidence: 68%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Contributions of Sinorhizobium meliloti Transcriptional Regulator DksA to Bacterial Growth and Efficient Symbiosis with Medicago sativa

Wippel

Long

2016

J Bacteriol

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“…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)(14)(15)(16) and its analog, DksA2, from Pseudomonas aeruginosa (17), as well as its structural homolog, TraR, often found on conjugative plasmids (18).…”

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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