scholarly journals Molecular mechanism of regulation of the purine salvage enzyme XPRT by the alarmones pppGpp, ppGpp, and pGpp

2020 ◽  
Author(s):  
Brent W. Anderson ◽  
Aili Hao ◽  
Kenneth A. Satyshur ◽  
James L. Keck ◽  
Jue D. Wang
Keyword(s):  
Binding Sites ◽  
Bacterial Species ◽  
Evolutionary Conservation ◽  
Differential Regulation ◽  
Hill Coefficient ◽  
Binding Motif ◽  
Salvage Pathway ◽  
Purine Salvage ◽  
Starvation Response ◽  
Dimer Interface

ABSTRACTThe alarmones pppGpp and ppGpp mediate starvation response and maintain purine homeostasis to protect bacterial species. Xanthine phosphoribosyltransferase (XPRT) is a purine salvage enzyme that produces the nucleotide XMP from PRPP and xanthine. Combining structural, biochemical and genetic analyses, we show that pppGpp and ppGpp, as well as a third putative alarmone pGpp, all directly interact with XPRT and inhibit XPRT activity by competing with its substrate PRPP. Structural analysis reveals that ppGpp binds the PRPP binding motif within the XPRT active site. This motif is present in another (p)ppGpp target, the purine salvage enzyme HPRT, suggesting evolutionary conservation in different enzymes. However, XPRT oligomeric interaction is distinct from HPRT in that XPRT forms a symmetric dimer with two (p)ppGpp binding sites at the dimer interface. This results in two distinct regulatory features. First, XPRT cooperatively binds (p)ppGpp with a Hill coefficient of 2. Also, XPRT displays differential regulation by the alarmones as it is potently inhibited by both ppGpp and pGpp, but only modestly by pppGpp. Lastly, we demonstrate that the alarmones are necessary for protecting GTP homeostasis against excess environmental xanthine in Bacillus subtilis, suggesting that regulation of XPRT is key for regulating the purine salvage pathway.

scholarly journals Mutational Analysis of the Flagellar Rotor Protein FliN: Identification of Surfaces Important for Flagellar Assembly and Switching

2006 ◽  
Vol 188 (14) ◽  
pp. 5240-5248 ◽  
Author(s):  
Koushik Paul ◽  
Jacob G. Harmon ◽  
David F. Blair
Keyword(s):  
Binding Sites ◽  
Mutational Analysis ◽  
Bacterial Species ◽  
Cross Linking ◽  
Binding Interaction ◽  
Dimer Interface ◽  
Hydrophobic Patch ◽  
Flagellar Assembly ◽  
And Function ◽  
Assembly Defect

ABSTRACT FliN is a component of the flagellar switch complex in many bacterial species. The crystal structure is known for most of FliN, and a targeted cross-linking study (K. Paul and D. F. Blair, J. Bacteriol. 188:2502-2511, 2006) showed that it is organized in ring-shaped tetramers at the bottom of the basal body C ring. FliN is essential for flagellar assembly and direction switching, but its precise functions have not been defined. Here, we identify functionally important regions on FliN by systematic mutagenesis. Nonconservative mutations were made at positions sampling the surface of the protein, and the effects on flagellar assembly and function were measured. Flagellar assembly was disrupted by mutations in a conserved hydrophobic patch centered on the dimer twofold axis or by mutations on the surface that forms the dimer-dimer interface in the tetramer. The assembly defect in hydrophobic-patch mutants was partially rescued by overexpression of the flagellar export proteins FliH and FliI, and coprecipitation assays demonstrated a binding interaction between FliN and FliH that was weakened by mutations in the hydrophobic patch. Thus, FliN might contribute to export by providing binding sites for FliH or FliH-containing complexes. The region around the hydrophobic patch is also important for switching; certain mutations in or near the patch caused a smooth-swimming chemotaxis defect that in most cases could be partially rescued by overexpression of the clockwise-signaling protein CheY. The results indicate that FliN is more closely involved in switching than has been supposed, possibly contributing to the binding site for CheY on the switch.

scholarly journals Effects of the Chromosome Partitioning Protein Spo0J (ParB) on oriC Positioning and Replication Initiation in Bacillus subtilis

2003 ◽  
Vol 185 (4) ◽  
pp. 1326-1337 ◽  
Author(s):  
Philina S. Lee ◽  
Daniel Chi-Hong Lin ◽  
Shigeki Moriya ◽  
Alan D. Grossman
Keyword(s):  
Bacillus Subtilis ◽  
Binding Sites ◽  
Fluorescent Protein ◽  
Bacterial Species ◽  
Significant Proportion ◽  
Subcellular Location ◽  
Replication Initiation ◽  
Null Mutant ◽  
Protein Ratio ◽  
Chromosome Partitioning

ABSTRACT Spo0J (ParB) of Bacillus subtilis is a DNA-binding protein that belongs to a conserved family of proteins required for efficient plasmid and chromosome partitioning in many bacterial species. We found that Spo0J contributes to the positioning of the chromosomal oriC region, but probably not by recruiting the origin regions to specific subcellular locations. In wild-type cells during exponential growth, duplicated origin regions were generally positioned around the cell quarters. In a spo0J null mutant, sister origin regions were often closer together, nearer to midcell. We found, by using a Spo0J-green fluorescent protein [GFP] fusion, that the subcellular location of Spo0J was a consequence of the chromosomal positions of the Spo0J binding sites. When an array of binding sites (parS sites) were inserted at various chromosomal locations in the absence of six of the eight known parS sites, Spo0J-GFP was no longer found predominantly at the cell quarters, indicating that Spo0J is not sufficient to recruit chromosomal parS sites to the cell quarters. spo0J also affected chromosome positioning during sporulation. A spo0J null mutant showed an increase in the number of cells with some origin-distal regions located in the forespore. In addition, a spo0J null mutation caused an increase in the number of foci per cell of LacI-GFP bound to arrays of lac operators inserted in various positions in the chromosome, including the origin region, an increase in the DNA-protein ratio, and an increase in origins per cell, as determined by flow cytometry. These results indicate that the spo0J mutant produced a significant proportion of cells with increased chromosome content, probably due to increased and asynchronous initiation of DNA replication.

scholarly journals Human organic anion transporter MRP4 (ABCC4) is an efflux pump for the purine end metabolite urate with multiple allosteric substrate binding sites

2005 ◽  
Vol 288 (2) ◽  
pp. F327-F333 ◽  
Author(s):  
Rémon A. M. H. Van Aubel ◽  
Pascal H. E. Smeets ◽  
Jeroen J. M. W. van den Heuvel ◽  
Frans G. M. Russel
Keyword(s):  
Binding Sites ◽  
Efflux Pump ◽  
Substrate Binding ◽  
Organic Anion ◽  
Apical Cell ◽  
Cell Uptake ◽  
Hek293 Cells ◽  
Hill Coefficient ◽  
Anion Transporter ◽  
Substrate Binding Sites

The end product of human purine metabolism is urate, which is produced primarily in the liver and excreted by the kidney through a well-defined basolateral blood-to-cell uptake step. However, the apical cell-to-urine efflux mechanism is as yet unidentified. Here, we show that the renal apical organic anion efflux transporter human multidrug resistance protein 4 (MRP4), but not apical MRP2, mediates ATP-dependent urate transport via a positive cooperative mechanism ( Km of 1.5 ± 0.3 mM, Vmax of 47 ± 7 pmol·mg−1·min−1, and Hill coefficient of 1.7 ± 0.2). In HEK293 cells overexpressing MRP4, intracellular urate levels were lower than in control cells. Urate inhibited methotrexate transport (IC50 of 235 ± 8 μM) by MRP4, did not affect cAMP transport, whereas cGMP transport was stimulated. Urate shifted cGMP transport by MRP4 from positive cooperativity ( Km and Vmax value of 180 ± 20 μM and 58 ± 4 pmol·mg−1·min−1, respectively, Hill coefficient of 1.4 ± 0.1) to single binding site kinetics ( Km and Vmax value of 2.2 ± 0.9 mM and 280 ± 50 pmol·mg−1·min−1, respectively). Finally, MRP4 could transport urate simultaneously with cAMP or cGMP. We conclude that human MRP4 is a unidirectional efflux pump for urate with multiple allosteric substrate binding sites. We propose MRP4 as a candidate transporter for urinary urate excretion and suggest that MRP4 may also mediate hepatic export of urate into the circulation, because of its basolateral expression in the liver.

scholarly journals Contingency in the convergent evolution of a regulatory network: Dosage compensation in Drosophila

2018 ◽  
Author(s):  
Doris Bachtrog ◽  
Chris Ellison
Keyword(s):  
Transposable Element ◽  
Dosage Compensation ◽  
Sex Chromosomes ◽  
Binding Sites ◽  
Evolutionary Biology ◽  
De Novo ◽  
Binding Motif ◽  
Sequence Motif ◽  
X Chromosomes ◽  
Msl Complex

The repeatability or predictability of evolution is a central question in evolutionary biology, and most often addressed in experimental evolution studies. Here, we infer how genetically heterogeneous natural systems acquire the same molecular changes, to address how genomic background affects adaptation in natural populations. In particular, we take advantage of independently formed neo-sex chromosomes in Drosophila species that have evolved dosage compensation by co-opting the dosage compensation (MSL) complex, to study the mutational paths that have led to the acquisition of 100s of novel binding sites for the MSL complex in different species. This complex recognizes a conserved 21-bp GA-rich sequence motif that is enriched on the X chromosome, and newly formed X chromosomes recruit the MSL complex by de novo acquisition of this binding motif. We identify recently formed sex chromosomes in the Drosophila repleta and robusta species groups by genome sequencing, and generate genomic occupancy maps of the MSL complex to infer the location of novel binding sites. We find that diverse mutational paths were utilized in each species to evolve 100s of de novo binding motifs along the neo-X, including expansions of microsatellites and transposable element insertions. However, the propensity to utilize a particular mutational path differs between independently formed X chromosomes, and appears to be contingent on genomic properties of that species, such as simple repeat or transposable element density. This establishes the “genomic environment” as an important determinant in predicting the outcome of evolutionary adaptations.

scholarly journals 3 minutes to precisely measure morphogen concentration

2018 ◽  
Author(s):  
Tanguy Lucas ◽  
Huy Tran ◽  
Carmina Angelica Perez Romero ◽  
Aurélien Guillou ◽  
Cécile Fradin ◽  
...  
Keyword(s):  
Binding Sites ◽  
Transcriptional Response ◽  
Positional Information ◽  
Fruit Fly ◽  
Fluorescent Labeling ◽  
Hill Coefficient ◽  
Activation Process ◽  
Endogenous Expression ◽  
Time Period ◽  
Zygotic Transcription

AbstractMorphogen gradients provide concentration-dependent positional information along polarity axes. Although the dynamics of establishment of these gradients is well described, precision and noise in the downstream activation processes remain elusive. A simple paradigm to address these questions is the Bicoid morphogen gradient that elicits a rapid step-like transcriptional response in young fruit fly embryos. Focusing on the expression of the main Bicoid target, hunchback (hb), at the onset of zygotic transcription, we used the MS2-MCP approach which combines fluorescent labeling of nascent mRNA with live imaging at high spatial and temporal resolution. Removing 36 putative Zelda binding sites unexpectedly present in the original MS2 reporter, we show that the 750 bp of the hb promoter are sufficient to recapitulate endogenous expression at the onset of zygotic transcription. After each mitosis, in the anterior, expression is turned on to rapidly reach a plateau with all nuclei expressing the reporter. Consistent with a Bicoid dose-dependent activation process, the time period required to reach the plateau increases with the distance to the anterior pole. Remarkably, despite the challenge imposed by frequent mitoses and high nuclei-to-nuclei variability in transcription kinetics, it only takes 3 minutes at each interphase for the MS2 reporter loci to measure subtle differences in Bicoid concentration and establish a steadily positioned and steep (Hill coefficient ~ 7) expression boundary. Modeling based on cooperativity between the 6 known Bicoid binding sites in the hb promoter region and assuming rate limiting concentrations of the Bicoid transcription factor at the boundary is able to capture the observed dynamics of pattern establishment but not the steepness of the boundary. This suggests that additional mechanisms are involved in the steepness of the response.

scholarly journals Similar solutions to a common challenge: Regulation of genes encoding Ralstonia solanacearum xanthine dehydrogenase

2021 ◽  
Author(s):  
Smitha Sivapragasam ◽  
Arpita Ghosh ◽  
Sanjay Kumar ◽  
Danté T Johnson ◽  
Anne Grove
Keyword(s):  
Transcription Factor ◽  
Ralstonia Solanacearum ◽  
Fluorescent Protein ◽  
Bacterial Species ◽  
Regulatory System ◽  
Stringent Response ◽  
Xanthine Dehydrogenase ◽  
Guanosine Monophosphate ◽  
Purine Salvage ◽  
Genes Encoding

Abstract The stringent response involves accumulation of (p)ppGpp, and it ensures that survival is prioritized. Production of (p)ppGpp requires purine synthesis, and upregulation of an operon that encodes the purine salvage enzyme xanthine dehydrogenase (Xdh) has been observed during stringent response in some bacterial species, where direct binding of ppGpp to a TetR-family transcription factor is responsible for increased xdh gene expression. We show here that the plant pathogen Ralstonia solanacearum has a regulatory system in which the LysR-family transcription factor XanR controls expression of the xan operon; this operon encodes Xdh as well as other enzymes involved in purine salvage, which favor accumulation of xanthine. XanR bound upstream of the xan operon, a binding that was attenuated on addition of either ppGpp or cyclic di-guanosine monophosphate (c-di-GMP). Using a reporter in which enhanced green fluorescent protein (EGFP) is expressed under control of a modified xan promoter, XanR was shown to repress EGFP production. Our data suggest that R. solanacearum features a regulatory mechanism in which expression of genes encoding purine salvage enzymes is controlled by a transcription factor that belongs to a different protein family, yet performs similar regulatory functions.

scholarly journals Purine nucleoside phosphorylase in chronic lymphocytic leukemia (CLL)

Blood ◽  
1978 ◽  
Vol 52 (5) ◽  
pp. 886-895 ◽  
Author(s):  
M Borgers ◽  
H Verhaegen ◽  
M De Brabander ◽  
J De Cree ◽  
W De Cock ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Healthy Subjects ◽  
Purine Nucleoside Phosphorylase ◽  
Peripheral Blood Lymphocytes ◽  
Lymphocytic Leukemia ◽  
Purine Nucleoside ◽  
Salvage Pathway ◽  
Purine Salvage ◽  
Nucleoside Phosphorylase ◽  
A Minor

Abstract Purine nucleoside phosphorylase (PNP), the enzyme schematically next to adenosine deaminase in the purine salvage pathway, has been demonstrated cytochemically in peripheral blood lymphocytes of healthy subjects and chronic lymphocytic leukemia (CLL) patients. The enzyme activity is confined to the cytosol. In healthy subjects the majority of lymphocytes are strongly reactive for PNP, whereas the rest are devoid of cytochemically demonstrable activity. The percentage of PNP- positive cells largely corresponds to the number of E rosette-forming cells and is inversely proportional to the number of Ig-bearing cells. In six of seven CLL patients studied only a minor percentage of the lymphocytes showed strong PNP activity, whereas the large majority (88%- -98%) possessed trace activity. Such patients have a high number of Ig- bearing cells and a low number of E rosette-forming cells. A different pattern of markers was found in the lymphocytes of the seventh CLL patient: 66% were strongly reactive for PNP, an important number formed E rosettes, and a minor percentage were Ig bearing. These data indicate that PNP can be useful as a “nonmembrane” marker in the differentiation of the B and T cell origin in CLL and deserves to be studied in other lymphoproliferative disorders.

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