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A new method for automatically assigning proton-proton NOESY spectra is described and demonstrated for simulated and experimental spectra of the proteins dendrotoxin K, alpha-amylase inhibitor tendamistat and the DNA-binding domain of the 434 repressor protein. The method assigns the NOESY spectrum and calculates three-dimensional protein structures simultaneously, using a list of proton chemical shifts and 3JNH alpha coupling constants. An ensemble of structures is iteratively calculated by self-correcting distance geometry from unambiguous and selected ambiguous NOESY cross peaks. New structure based filters recognize the correct constraints from the ambiguous cross peak list. For the first round of assignment neither a preliminary initial structure nor a sufficient set of unambiguous NOESY cross peaks is needed. The method can also be applied to cross peak lists containing hundreds of noise peaks. For an assumed tolerance of +/- 0.01 ppm in the chemical shifts of the peak positions, only about 10% of the NOESY cross peaks can be unambiguously assigned based on their chemical shifts alone. Our automated method assigned about 80% of all cross peaks with this chemical shift tolerance, and 95 to 99% of the assignments were correct. The average pairwise RMSD for the backbone atoms of the ten best final structures is about 1.5 A in all three proteins and the previously determined NMR solution structures are always embedded in this structure bundle. We regard our method as a highly practical tool for automatic calculation of three-dimensional protein structures from NMR spectra with minimal human interference.
J Mol Biol 1995 Dec 01
PMID:Automated assignment of simulated and experimental NOESY spectra of proteins by feedback filtering and self-correcting distance geometry. 749 Jul 63

The fine-control of gene expression in the trp repressor system is achieved through the thermodynamic linkage of multiple equilibria involving the trp repressor protein (TR), tryptophan (L-Trp) and DNA. We have undertaken studies of superrepressor mutants of TR as a means of dissecting the coupled equilibria that contribute to repressor function. Unlike all the other tested super-repressors that exhibit differences from wild-type TR DNA binding affinity or stoichiometry, the AV77 superrepressor (an alanine to valine substitution at position 77: AV77TR) has been indistinguishable from TR in vitro. The present studies using a variety of biophysical measurements comparing TR and AV77TR provide strong evidence that the helix-turn-helix (HTH) region of apoTR exists in a partially folded conformation. Far UV CD spectra of the two proteins reveal a 10% increase in helical content for the apoAV77TR compared to apoTR. Moreover, urea denaturation studies demonstrate that apoAV77TR is more stable to denaturation than apoTR. ApoTR binds large amounts of 1,8-ANS, a hydrophobic fluorescence probe used to detect protein folding intermediates, with high affinity, where apoAV77TR exhibits only marginal binding of this ligand. While the tryptophan affinities of the two proteins as measured by titration calorimetry are quite similar, the thermodynamic signatures are distinct, with a much reduced unfavorable entropic contribution for AV77TR. Finally, the allosteric effect of L-Trp on oligomerization is abolished by the AV77 mutation. Taken together these data support previous calorimetric studies implicating coupling of folding and L-Trp binding for TR. Moreover, they are consistent with NMR observations indicating partial disorder in the HTH region of apoTR. Based upon the distinct biophysical properties of TR and AV77TR, we propose a model in which folding of the HTH region accompanies ligand binding in TR. In this model distinct protein-protein interactions of the apo- and holoTR link this conformational change to apparent operator affinities, thereby modulating TR function in vivo.
J Mol Biol 1995 Oct 20
PMID:Evidence for coupling of folding and function in trp repressor: physical characterization of the superrepressor mutant AV77. 756 88

We describe the construction and analysis of derivatives of the yeast TDH3 promoter in which the TATA box element has been replaced by a portion of the phage lambda operator containing a consensus TATA site flanked by binding sites for the cI repressor. Transcription of a reporter gene under the control of such a promoter is reduced in cells that express the cI repressor protein. Deletion of the native TATA element of the TDH3 promoter reduces transcription to the same extent. The cI repressor may act by "masking" the TATA element located between the repressor binding sites. Furthermore, the use of a temperature-sensitive cI repressor allowed temperature-dependent transcription of the reporter gene.
Mol Gen Genet 1995 Aug 30
PMID:A temperature-sensitive lambda cI repressor functions on a modified operator in yeast cells by masking the TATA element. 756 15

We present a procedure for automatically identifying from a set of aligned protein structures a subset of atoms with only a small amount of structural variation, i.e., a core. We apply this procedure to the globin family of proteins. Based purely on the results of the procedure, we show that the globin fold can be divided into two parts. The part with greater structural variation consists of the residues near the heme (the F helix and parts of the G and H helices), and the part with lesser structural variation (the core) forms a structural framework similar to that of the repressor protein (A, B, and E helices and remainder of the G and H helices). Such a division is consistent with many other structural and biochemical findings. In addition, we find further partitions within the core that may have biological significance. Finally, using the structural core of the globin family as a reference point, we have compared structural variation to sequence variation and shown that a core definition based on sequence conservation does not necessarily agree with one based on structural similarity.
Proc Int Conf Intell Syst Mol Biol 1994
PMID:Finding an average core structure: application to the globins. 758 90

Semi-dominant mutations in the amdA gene lead to elevated expression of the gene encoding acetamidase, amdS. These mutations also cause constitutive expression of the acetate-inducible gene, aciA. In the amdS 5' regulatory region, two cis-acting mutations, amdl66 and amdl666, have been isolated which specifically affect amdA activation of amdS. These mutations are a duplication and a triplication of an 18 bp GA-rich sequence, thought to define the amdA site of action within the amdS promoter region. Similar GA-rich sequences have also been found in the 5' region of aciA. This paper describes the cloning and initial functional characterization of the amdA gene and two of its mutant alleles. The wild-type amdA gene has been cloned by a chromosome walk from genes gatA and alcC on linkage group VII and localized by complementation of an amdA loss-of-function mutation. Transcriptional analysis reveals that the gene is expressed constitutively at low levels under growth conditions which affect expression of amdS and aciA. The gene is predicted to encode an 880-amino-acid protein which contains two C2H2 zinc fingers, a nuclear localization sequence and two transcriptional activation domains. The amdA7 semi-dominant gain-of-function mutation results in a glycine to aspartate substitution which would increase the acidity of one of these regions. Analysis of in vitro generated mutations in the 5' region of amdS using an amdS::lacZ reporter has been used to localize the site of action of AmdA. The C2H2 zinc-finger motifs identified in the protein are similar to those found in the carbon catabolite repressor protein, CreA, which also regulates amdS and recognizes sequences which overlap with the proposed site of action for AmdA.
Mol Microbiol 1995 Mar
PMID:The positively acting amdA gene of Aspergillus nidulans encodes a protein with two C2H2 zinc-finger motifs. 759 97

Pseudobactin 358 is the yellow-green fluorescent siderophore produced by Pseudomonas putida WCS358 in conditions of iron limitation. The genes encoding for siderophore biosynthesis are iron-regulated at the transcriptional level. Previous work has shown that a positive regulator, PfrA, is absolutely required for the activation under iron-limiting conditions of pseudobactin 358 biosynthesis. In this study we identified a set of Tn5 insertion mutants of strain WCS358 which lost the ability to activate an iron-regulated siderophore promoter. These mutants no longer produced pseudobactin 358. Molecular analysis revealed that they carried a Tn5 insertion in a gene, designated pfrl (Pseudomonas ferric regulator), which codes for a protein (Pfrl) of 19.5 kDa. Pfrl contains a putative helix-turn-helix motif typical of DNA-binding proteins and has homology to two DNA-binding transcriptional activators, Fecl from Escherichia coli and Pupl from P. putida. The proposed role of Pfrl in strain WCS358 is an activator protein regulating pseudobactin 358 biosynthesis under iron limitation. The pfrl promoter region contains a sequence which displays high identity to the Fur-box consensus. This 19 bp consensus sequence is recognized by Fur, an iron-binding repressor protein found in many different bacteria. The E. coli Fur protein can bind to the pfrl promoter region, indicating that this activator gene is likely to be iron-regulated by Fur. We also report the identification and characterization of the P. putida WCS358 fur gene. The Fur protein of strain WCS358 is structurally and functionally similar to other cloned Fur proteins from other bacterial species.
Mol Microbiol 1995 Mar
PMID:Iron regulation of siderophore biosynthesis and transport in Pseudomonas putida WCS358: involvement of a transcriptional activator and of the Fur protein. 762 64

The rot gene in Escherichia coli encodes PPIase A, a periplasmic peptidyl-prolyl cis-trans isomerase with homology to the cyclophilin family of proteins. Here it is demonstrated that rot is expressed in a complex manner from four overlapping promoters and that the rot regulatory region is unusually compact, containing a close array of sites for DNA-binding proteins. The three most upstream rot promoters are activated by the global gene regulatory cAMP-CRP complex and negatively regulated by the CytR repressor protein. Activation of these three promoters occurs by binding of cAMP-CRP to two sites separated by 53 bp. Moreover, one of the cAMP-CRP complexes is involved in the activation of both a Class I and a Class II promoter. Repression takes place by the formation of a CytR/cAMP-CRP/DNA nucleoprotein complex consisting of the two cAMP-CRP molecules and CytR bound in between. The two regulators bind co-operatively to the DNA overlapping the three upstream promoters, simultaneously quenching the cAMP-CRP activator function. These results expand the CytR regulon to include a gene whose product has no known function in ribo- and deoxyribonucleoside catabolism or transport.
Mol Microbiol 1994 Dec
PMID:The gene encoding the periplasmic cyclophilin homologue, PPIase A, in Escherichia coli, is expressed from four promoters, three of which are activated by the cAMP-CRP complex and negatively regulated by the CytR repressor. 771 59

We used a one-hybrid system to replace precisely the finger II chicken GATA-1 DNA-binding domain with the binding domain of bacterial repressor protein LexA. The LexA DNA-binding domain lacks amino acids that function for transcriptional activation, nuclear localization, or protein dimerization. This allowed us to analyze activities of GATA-1 sequences distinct from DNA binding. We found that strong transcriptional activating sequences that function independently of finger II are present in GATA-1. Sequences including finger I contain an independent nuclear localizing function. Our data are consistent with cooperative binding of two LexA-GATA-1 hybrid proteins on a palindromic operator. The sensitivity of our transcription assay provides the first evidence that GATA-1 can make homotypic interactions in vivo. The ability of a non-DNA-binding form of GATA-1 to activate gene expression by targeting to a bound GATA-1 derivative further supports the notion that GATA-1-GATA-1 interactions may have functional consequences. A coimmunoprecipitation assay was used to demonstrate that GATA-1 multimeric complexes form in solution by protein-protein interaction. The novel ability of GATA-1 to interact homotypically may be important for the formation of higher-order structures among distant regulatory elements that share binding sites for this transcription factor. We also used the system to test the ability of GATA-1 to interact heterotypically with other activators.
Mol Cell Biol 1995 Mar
PMID:Homotypic interactions of chicken GATA-1 can mediate transcriptional activation. 786 28

Many bacteria respond to a lack of iron in the environment by synthesizing siderophores, which act as iron-scavenging compounds. Fluorescent pseudomonads synthesize strain-specific but chemically related siderophores called pyoverdines or pseudobactins. We have investigated the mechanisms by which iron controls expression of genes involved in pyoverdine metabolism in Pseudomonas aeruginosa. Transcription of these genes is repressed by the presence of iron in the growth medium. Three promoters from these genes were cloned and the activities of the promoters were dependent on the amounts of iron in the growth media. Two of the promoters were sequenced and the transcriptional start site were identified by S1 nuclease analysis. Sequences similar to the consensus binding site for the Fur repressor protein, which controls expression of iron-repressible genes in several gram-negative species, were not present in the promoters, suggesting that they are unlikely to have a high affinity for Fur. However, comparison of the promoter sequences with those of iron-regulated genes from other Pseudomonas species and also the iron-regulated exotoxin gene of P. aeruginosa allowed identification of a shared sequence element, with the consensus sequence (G/C)CTAAAT-CCC, which is likely to act as a binding site for a transcriptional activator protein. Mutations in this sequence greatly reduced the activities of the promoters characterized here as well as those of other iron-regulated promoters. The requirement for this motif in the promoters of iron-regulated genes of different Pseudomonas species indicates that similar mechanisms are likely to be involved in controlling expression of a range of iron-regulated genes in pseudomonads.
Mol Gen Genet 1995 Feb 20
PMID:Identification of a DNA sequence motif required for expression of iron-regulated genes in pseudomonads. 789 66

The iron-repressible outer membrane protein FyuA of Yersinia enterocolitica operates as a receptor with dual function: (i) as a receptor for the Y. pestis bacteriocin pesticin, and (ii) as a receptor for yersiniabactin, a siderophore that is produced by mouse-virulent Y. enterocolitica strains of biogroup IB. Cloning of the FyuA-encoding gene was achieved by mobilization of a genomic cosmid library of the pesticin-sensitive and mouse-virulent Y. enterocolitica O:8 strain WA into the pesticin-resistant WA fyuA mutant and subsequent in vivo selection of transconjugants for the ability to survive and multiply in mice (phenotype mouse virulence). The reisolated transconjugants which survived in mice for 3 d harboured a unique cosmid and phenotypically were pesticin sensitive. From this cosmid a 2650 bp SalI-PstI fragment conferring pesticin sensitivity was subcloned. Sequencing of this DNA fragment revealed a single open reading frame of 2022 bp, which encodes a deduced polypeptide of 673 amino acids with a predicted molecular mass of 73,677 Da. Cleavage of a putative signal sequence composed of 22 amino acids should lead to a mature protein of 651 amino acids with a molecular mass of 71,368 Da. The open reading frame is preceded by a sequence which shares homology with the postulated consensus Fur iron-repressor protein-binding site. FyuA shows homology to other iron-regulated TonB-dependent outer membrane proteins with receptor functions (e.g. BtuB, CirA, FepA, IutA, FhuA, FoxA, FcuA). On the basis of multiple alignment of amino acid sequences of FyuA and other TonB-dependent receptors, a phylogenetic tree was constructed, demonstrating that FyuA probably belongs to the citrate subfamily or represents a new subfamily of TonB-dependent receptors. Moreover, by complementation of the WA fyuA mutant by the cloned fyuA gene, yersiniabactin uptake and mouse virulence were restored. These studies demonstrate that the cloned pesticin/yersiniabactin receptor FyuA of Y. enterocolitica has the typical features of iron-regulated TonB-dependent outer membrane receptors for siderophores and bacteriocins and is required for mouse virulence.
Mol Microbiol 1994 Jul
PMID:The pesticin receptor of Yersinia enterocolitica: a novel virulence factor with dual function. 798 5


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