The solution structure of the Leu22 → Val mutant AREA DNA binding domain complexed with a TGATAG core element defines a role for hydrophobic packing in the determination of specificity 1 1Edited by P. E. Wright

Starich, Mary R.; Wikström, Mats; Schumacher, S.; Arst, Herbert N.; Gronenborn, Angela M.; Clore, G. Marius

doi:10.1006/jmbi.1997.1626

Cited by 37 publications

(27 citation statements)

References 24 publications

(59 reference statements)

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“…This strain expresses a Zn-finger mutant of WC-2 in which two basic residues implicated in DNA binding were replaced by acidic residues ( Fig. 1G; Starich et al 1998). WC-2 RK/DD did not support expression of FRQ ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Circadian activity and abundance rhythms of the Neurospora clock transcription factor WCC associated with rapid nucleo–cytoplasmic shuttling

Schafmeier

Diernfellner²,

Schäfer³

et al. 2008

Genes Dev.

122

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The Neurospora clock protein FREQUENCY (FRQ) inhibits its transcriptional activator WHITE COLLAR COMPLEX (WCC) in a negative feedback loop and supports its accumulation in a positive loop. We show that positive feedback is a delayed effect of negative feedback underlying the same post-translational mechanisms: DNA-binding-competent active WCC commits rapidly to degradation. FRQ-dependent phosphorylation of WCC, which interferes with DNA binding (negative feedback), leads to reduced turnover and slow accumulation of newly expressed WCC (positive feedback). When DNA binding of WCC is compromised by mutation, its accumulation is independent of FRQ. Cycles of FRQ-dependent inactivation and PP2A-dependent reactivation of WCC occur in the minute range and are coupled to obligate rapid cycles of nucleo-cytoplasmic shuttling. WCC shuttling and activity cycles are modulated by FRQ in circadian fashion. We show here that FRQ supports negative and positive limbs of the clock by the same molecular mechanisms. Positive feedback (FRQ-dependent accumulation of WCC) is a delayed consequence of negative feedback (FRQ-dependent inactivation of WCC) rather than a mechanistically distinct feedback loop: WCC is active when FRQ is low or absent. Our data indicate that DNAbinding-competent, active WCC is unstable and rapidly turned over. FRQ-dependent phosphorylation of WCC interferes with DNA binding. This results in reduced turnover and allows accumulation of newly expressed WCC. Inactivation and reactivation of WCC are coupled to cycles of nucleo-cytoplasmic shuttling. We show that PP2A/RGB-1 activity is cytoplasmic, and hence passage of the WCC through the cytosol is obligatory for reactivation. Surprisingly, phosphorylation and shuttling cycles occur in the range of minutes and are modulated by FRQ in circadian fashion. Results and DiscussionWe investigated whether FRQ affects turnover of the WCC. In wild type, WCC is stable in constant darkness (DD) but turned over rapidly in constant light (LL) (Lee et al. 2000). To assess the influence of FRQ on WCC turnover, cultures of wild type and frq 9 , a mutant strain harboring a nonfunctional frq allele, were grown in LL. Turnover kinetics were then measured in the presence of cycloheximide (CHX). Degradation of WCC was substantially faster in frq 9 (t 1/2 ∼ 2.4 h) than in wild type (t 1/2 ∼ 4.2 h), demonstrating that FRQ stabilizes the light-activated WCC (Fig. 1A,F).In the negative feedback loop, FRQ promotes phosphorylation of WCC, which leads to its inactivation (Schafmeier et al. 2005). To investigate whether WCC activity affects its stability, we analyzed turnover of WCC in the wc-2G3 strain (Linden et al. 1997). wc-2G3 encodes a WC-2 version that lacks the C-terminal Zincfinger (Zn-finger) domain (Fig. 1B) and is henceforth referred to as wc2⌬C. WC-1 is unstable and does not accumulate in the absence of its assembly partner WC-2 (Cheng et al. 2002). WC-1 accumulated in high levels in wc-2⌬C (Fig. 1C), demonstrating that it assembled with the truncated WC-2⌬C. However, the mutan...

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Section: Resultsmentioning

confidence: 99%

Circadian activity and abundance rhythms of the Neurospora clock transcription factor WCC associated with rapid nucleo–cytoplasmic shuttling

Schafmeier

Diernfellner²,

Schäfer³

et al. 2008

Genes Dev.

122

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“…Like Gln3, the AREA and cGATA-1 proteins are GATA-binding proteins (Orkin 1992;Merika and Orkin 1993;Ko and Engel 1993;Ravagnani et al 1997;Scazzocchio 2000). To our advantage, 3D structures of AREA and cGATA-1 in complexes with their GATA DNA target sites have been determined using NMR methods (Merika and Orkin 1993;Omichinski et al 1993a,b;Stahl and Gronenborn 1993;Starich et al 1998;Kotaka et al 2008;Scazzocchio 2000).…”

Section: Identification Of a Gln3 Sequence With The Characteristics Omentioning

confidence: 99%

Nuclear Gln3 Import Is Regulated by Nitrogen Catabolite Repression Whereas Export Is Specifically Regulated by Glutamine

et al. 2015

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Gln3, a transcription activator mediating nitrogen-responsive gene expression in Saccharomyces cerevisiae, is sequestered in the cytoplasm, thereby minimizing nitrogen catabolite repression (NCR)-sensitive transcription when cells are grown in nitrogen-rich environments. In the face of adverse nitrogen supplies, Gln3 relocates to the nucleus and activates transcription of the NCR-sensitive regulon whose products transport and degrade a variety of poorly used nitrogen sources, thus expanding the cell's nitrogen-acquisition capability. Rapamycin also elicits nuclear Gln3 localization, implicating Target-of-rapamycin Complex 1 (TorC1) in nitrogen-responsive Gln3 regulation. However, we long ago established that TorC1 was not the sole regulatory system through which nitrogen-responsive regulation is achieved. Here we demonstrate two different ways in which intracellular Gln3 localization is regulated. Nuclear Gln3 entry is regulated by the cell's overall nitrogen supply, i.e., by NCR, as long accepted. However, once within the nucleus, Gln3 can follow one of two courses depending on the glutamine levels themselves or a metabolite directly related to glutamine. When glutamine levels are high, e.g., glutamine or ammonia as the sole nitrogen source or addition of glutamine analogues, Gln3 can exit from the nucleus without binding to DNA. In contrast, when glutamine levels are lowered, e.g., adding additional nitrogen sources to glutamine-grown cells or providing repressive nonglutamine nitrogen sources, Gln3 export does not occur in the absence of DNA binding. We also demonstrate that Gln3 residues 64-73 are required for nuclear Gln3 export.

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“…areA mutations in the DNA-binding domain that differentially affect the expression of a number of genes have been described. Genetic, footprinting, modeling and nuclear magnetic resonance studies have shown that this is due to differential binding to different GATA sites within the HGATAR consensus (6,31,50,59,60,65).…”

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confidence: 99%

A Paradoxical Mutant GATA Factor

Muro‐Pastor¹,

Strauss

Ramón³

et al. 2004

Eukaryot Cell

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The niiA (nitrite reductase) and niaD (nitrate reductase) genes of Aspergillus nidulans are subject to both induction by nitrate and repression by ammonium or glutamine. The intergenic region between these genes functions as a bidirectional promoter. In this region, nucleosomes are positioned under nonexpression conditions. On nitrate induction under derepressing conditions, total loss of positioning occurs. This is independent of transcription and of the NirA-specific transcription factor but absolutely dependent on the widedomain GATA-binding AreA factor. We show here that a 3-amino-acid deletion in the basic carboxy-terminal sequence of the DNA-binding domain results in a protein with paradoxical properties. Its weak DNA binding is consistent with its loss-of-function phenotype on most nitrogen sources. However, it results in constitutive expression and superinducibility of niiA and niaD. Nucleosome loss of positioning is also constitutive. The mutation partially suppresses null mutations in the transcription factor NirA. AreA binds NirA in vitro, and the mutation does not affect this interaction. The in vivo methylation pattern of the promoter is drastically altered, suggesting the recruitment of one or more unknown transcription factors and/or a local distortion on the DNA double helix.The transcriptional activation of the niiA (nitrite reductase) and niaD (nitrate reductase) genes of Aspergillus nidulans involves a 1.2-kb intergenic region. The activation process is strictly dependent on two transcription factors, NirA, a Zn binuclear cluster protein, and AreA, a GATA factor (7,41,48,61) NirA is specific for the nitrate assimilation pathway, while AreA is involved in the transcriptional activation of scores of genes involved in the utilisation of different nitrogen sources (see references 4, 35, and 51 for reviews). Activation of transcription has an absolute requirement for both the presence of the specific inducer (nitrate) and the absence of repressing nitrogen metabolites (ammonium and glutamine [41]). The standard model is that AreA mediates general derepression in the absence of ammonium or glutamine while NirA mediates specific induction by nitrate (7,39). Results presented here and in reference 41 make this simple model untenable. A very similar system of control is extant in Neurospora crassa, where close homologues of NirA and AreA operate (39), and probably in all filamentous ascomycetes able to assimilate nitrate (see, for example, references 18 and 19).

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The solution structure of the Leu22 → Val mutant AREA DNA binding domain complexed with a TGATAG core element defines a role for hydrophobic packing in the determination of specificity 1 1Edited by P. E. Wright

Cited by 37 publications

References 24 publications

Circadian activity and abundance rhythms of the Neurospora clock transcription factor WCC associated with rapid nucleo–cytoplasmic shuttling

Circadian activity and abundance rhythms of the Neurospora clock transcription factor WCC associated with rapid nucleo–cytoplasmic shuttling

Nuclear Gln3 Import Is Regulated by Nitrogen Catabolite Repression Whereas Export Is Specifically Regulated by Glutamine

A Paradoxical Mutant GATA Factor

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