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In this study, we examined how the Drosophila developmental control gene even-skipped (eve) represses transcription. Tissue culture cells were used to show that eve contains domains which inhibit transcriptional activators present at the Ultrabithorax (Ubx) proximal promoter when bound up to 1.5 kb away from these activators. Different portions of eve were fused to a heterologous DNA binding domain to show that three adjacent regions of eve contribute to silencing. There appear to be two mechanisms by which eve protein represses transcription. In this study, we used in vitro transcription and DNA binding experiments to provide evidence for one of these mechanisms. Repression in vitro correlates with binding of eve protein to two low-affinity sites in the Ubx proximal promoter. Occupancy of these low-affinity sites is dependent upon cooperative binding of other eve molecules to a separate high-affinity site. Some of these sites are separated by over 150 bp of DNA, and the data suggest that this intervening DNA is bent to form a looped structure similar to those caused by prokaryotic repressors. One of the low-affinity sites overlaps an activator element bound by the zeste transcription factor. Binding of eve protein is shown to exclude binding by zeste protein. These data suggest a mechanism for silencing whereby a repressor protein would be targeted to DNA by a high-affinity element, which itself does not overlap activator elements. Cooperative binding of further repressor molecules to distant low-affinity sites, and competition with activators bound at these sites lead to repression at a distance.
Mol Cell Biol 1993 May
PMID:Cooperative binding at a distance by even-skipped protein correlates with repression and suggests a mechanism of silencing. 809 76

In this paper we present a self-consistent ensemble optimization (SCEO) theory for efficient conformational search, which we have applied to predicting the effects of mutations on protein thermostability. This approach takes advantage of a statistical mechanical self-consistency condition to home in iteratively on the global minimum structure. We employ a fast potential of mean-force approximation to cut computation time to a few minutes for a typical protein mutation, with only linear time-dependence on the size of the prediction problem. Rather than seeking a single, static structure of minimum energy, the new method optimizes an ensemble of many conformations, seeking to predict the most likely ensemble for the native state at a desired temperature. Testing this approach with a simple physical model focusing entirely on steric interactions and side-chain rearrangement, we obtain robustly convergent prediction of core side-chain conformation, and of hydrophobic core mutations' effects on protein stability. Self-consistent ensemble optimization is superior to simulated annealing in its speed and convergence to the global minimum, and insensitive to starting conformation. In calculations on lambda repressor protein, structural predictions for an eight-residue molten-zone had side-chain r.m.s. error of 0.49 A for the wild-type protein. Evaluation of the method's mutant structure predictions should become possible, as structures of these mutant repressors are solved. Predicted energies for a series of nine hydrophobic core mutants correlated with measured free energies of unfolding with a coefficient of 0.82.
J Mol Biol 1994 Feb 25
PMID:Predicting protein mutant energetics by self-consistent ensemble optimization. 811 2

The regulation of hutA, the Vibrio cholerae gene encoding a 77-kDa iron-regulated outer membrane protein required for heme iron utilization, was characterized, and the DNA sequence of the gene was determined. A hutA::Tn5 lac fusion generated previously (D. P. Henderson and S. M. Payne, Mol. Microbiol. 7:461-469, 1993) was transformed into Fur- and Fur+ strains of Escherichia coli and V. cholerae. The results of beta-galactosidase assays on the transformed strains demonstrated that transcription of hutA is regulated by the Fur repressor protein in E. coli and at least partially regulated by Fur in V. cholerae. Analysis of the DNA sequence of hutA indicated that a sequence homologous to the E. coli consensus Fur box was present in the promoter region of hutA. The amino acid sequence of HutA is homologous to those of several TonB-dependent outer member proteins. However, when the V. cholerae heme utilization system, which requires one or more genes encoded by the recombinant plasmid pHUT10 in addition to hutA carried on a second vector, was transferred to a wild-type strain and an isogenic tonB mutant of E. coli, the tonB mutant could utilize heme iron as efficiently as the wild-type strain. These data indicate that the V. cholerae heme utilization system reconstituted in E. coli does not require a functional TonB protein. The tonB mutant transformed with the heme utilization plasmids could not utilize the siderophore ferrichrome as an iron source, indicating that none of the genes encoded on the heme utilization plasmids complements the tonB defect in E. coli. It is possible that a gene(s) encoded by the recombinant heme utilization plasmids encodes a protein serving a TonB-like function in V. cholerae. A region in the carboxy terminus of HutA is homologous to the horse hemoglobin gamma chain, and the amino acids involved in forming the heme pocket in the gamma chain are conserved in HutA. These data suggest that this region of HutA is involved in heme binding.
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PMID:Characterization of the Vibrio cholerae outer membrane heme transport protein HutA: sequence of the gene, regulation of expression, and homology to the family of TonB-dependent proteins. 819 82

The mtrR gene of Neisseria gonorrhoeae controls the level of susceptibility to hydrophobic antibiotics and detergents. The mtrR gene was cloned and shown to encode a putative transcriptional repressor. The mtr region was homologous to the envCD and acrAB regions of Escherichia coli, which are also involved in susceptibility to hydrophobic compounds. A homologous repressor protein was encoded by a previously unrecognized open reading frame within both the envCD and acrAB regions. Deletion of mtrR resulted in increased resistance to antibiotics and detergents: the mtrR mutations in two penicillin-resistant clinical isolates resulted in a change of His-105 to Tyr. We propose that the mtrR repressor allows gonococci to regulate the permeability of its cell envelope in response to environmental signals, so that they can grow in the presence of toxic faecal lipids in the rectum as well as in the genital tract.
Mol Microbiol 1994 Feb
PMID:Regulation of the permeability of the gonococcal cell envelope by the mtr system. 819 48

The plasmid-encoded arsenical resistance (ars) operon produces resistance to trivalent and pentavalent salts of arsenic and antimony. The first gene in the operon, arsR, was previously shown to encode a repressor protein. A newly identified gene, arsD, is shown here to encode a regulatory protein, the ArsD protein. The gene was identified by construction of an in-frame fusion between the C-terminally truncated arsD gene and the coding region for the mature form of beta-lactamase (blaM). The native arsD gene product was overexpressed and radioactively labelled as a 13 kDa polypeptide. A frameshift mutation within the arsD gene resulted in elevated levels of expression of downstream ars genes. Co-expression of a wild-type arsD gene in trans with the operon containing the mutated arsD gene reduced expression of the downstream genes to wild-type levels. The presence of the arsD gene had no effect on the basal level of operon expression set by the arsR gene product, and the repression produced by the arsD gene product was not affected by inducers of the operon. The results indicate that the ArsD protein is an inducer-independent trans-acting regulatory protein.
Mol Microbiol 1993 May
PMID:The arsD gene encodes a second trans-acting regulatory protein of the plasmid-encoded arsenical resistance operon. 832 69

The long terminal repeat (LTR) of the mouse mammary tumor virus was used as a template to examine the dual binding parameters of the glucocorticoid-receptor (GR) and a repressor protein termed Inhibitory Factor 1 (IF1). The receptor binds specifically to the glucocorticoid response element and precludes the binding of IF1 to its juxtaposed binding site within the LTR. When the two DNA targets are separated by the insertion of an additional 52 base pairs, coincident binding of both proteins is observed. Gel retention assays reveal three distinct nucleoprotein complexes. The first complex consists of the receptor and the LTR, the second is comprised of IF1 and DNA and the third is a multiprotein-DNA complex consisting of the GR, IF1 and DNA, migrating at a higher molecular weight position. The inhibition of IF1 binding by the presence of prebound GR leads to the repression of transcription of juxtaposed genes. The GR may act to block access of a sequence, used by the cell to titrate repressor proteins and facilitate the onset of gene expression.
Mol Cell Biochem 1993 May 12
PMID:The glucocorticoid receptor precludes the binding of a transcriptional repressor protein to the long terminal repeat of the mouse mammary tumor virus. 839 5

The diphtheria toxin repressor (DtxR) is an Fe(2+)-activated protein with sequence-specific DNA-binding activity for the diphtheria toxin (tox) operator. Under high-iron conditions in Corynebacterium diphtheriae, DtxR represses toxin and siderophore biosynthesis as well as iron uptake. DtxR and a mutant repressor with His-47 substituted for Arg-47, designated DtxR-R47H, were purified and compared. Six different divalent cations (Cd2+, Co2+, Fe2+, Mn2+, Ni2+, and Zn2+) activated the sequence-specific DNA-binding activity of DtxR and enabled it to protect the tox operator from DNase I digestion, but Cu2+ failed to activate DtxR. Hydroxyl radical footprinting experiments indicated that DtxR binds symmetrically about the dyad axis of the tox operator. Methylation protection experiments demonstrated that DtxR binding alters the susceptibility to methylation of three G residues within the AT-rich tox operator. These findings suggest that two or more monomers of DtxR are involved in binding to the tox operator, with symmetrical DNA-protein interactions occurring at each end of the palindromic operator. In this regard, DtxR resembles several other well-characterized prokaryotic repressor proteins but differs dramatically from the Fe(2+)-activated ferric uptake repressor protein (Fur) of Escherichia coli. The concentration of Co2+ required to activate DtxR-R47H was at least 10-fold greater than that needed to activate DtxR, but the sequence-specific DNA binding of activated DtxR-R47H was indistinguishable from that of wild-type DtxR. The markedly deficient repressor activity of DtxR-R47H is consistent with a significant decrease in its binding activity for divalent cations.
Mol Microbiol 1993 Jul
PMID:Analysis of diphtheria toxin repressor-operator interactions and characterization of a mutant repressor with decreased binding activity for divalent metals. 841 63

The scr regulon of pUR400 and the chromosomally encoded scr regulon of Klebsiella pneumoniae KAY2026 are both negatively controlled by a specific repressor (ScrR). As deduced from the nucleotide sequences, both scrR genes encode polypeptides of 334 residues (85.5% identical base pairs, 91.3% identical amino acids), containing an N-terminal helix-turn-helix motif. Comparison with other regulatory proteins revealed 30.6% identical amino acids to FruR, 27.0% to Lacl and 28.1% to GalR. Six scrRs super-repressor mutations define the inducer-binding domain. The scr operator sequences were identified by in vivo titration tests of the sucrose repressor and by in vitro electrophoretic mobility shift assays. D-fructose, an intracellular product of sucrose transport and hydrolysis, and D-fructose 1-phosphate were shown to be molecular inducers of both scr regulons. An active ScrR-FruR hybrid repressor protein was constructed with the N-terminal part of the sucrose repressor of K. pneumoniae and the C-terminal part of the fructose repressor of Salmonella typhimurium LT2. Gel retardation assays showed that the hybrid protein bound to scr-specific operators, and that D-fructose 1-phosphate, the inducer for FruR, was the only inducer. In vivo, neither the operators of the fru operon nor of the pps operon, the natural targets for FruR, were recognized, but the scr operators were. These data and the data obtained from the super-repressor alleles confirm previous models on the binding of repressors of the Lacl family to their operators.
Mol Microbiol 1993 Jul
PMID:Molecular analysis of two ScrR repressors and of a ScrR-FruR hybrid repressor for sucrose and D-fructose specific regulons from enteric bacteria. 841 65

The expression of functional T cell receptor-beta (TCR-beta) transcripts requires the activation of programmed DNA rearrangement events. It is not clear whether other mechanisms dictate TCR-beta mRNA levels during thymic ontogeny. We examined the potential role of RNA splicing as a regulatory mechanism. As a model system, we used an immature T cell clone, SL12.4, that transcribes a fully rearranged TCR-beta gene but essentially lacks mature 1.3-kb TCR-beta transcripts in the cytoplasm. Abundant TCR-beta splicing intermediates accumulate in the nucleus of this cell clone. These splicing intermediates result from inefficient or inhibited excision of four of the five TCR-beta introns; the only intron that is efficiently spliced is the most 5' intron, IVSL. The focal point for the regulation appears to be IVS1C beta 1 and IVS2C beta 1, since unusual splicing intermediates that have cleaved the 5' splice site but not the 3' splice site of these two introns accumulate in vivo. The block in 3' splice site cleavage is of interest since sequence analysis reveals that these two introns possess canonical splice sites. A repressional mechanism involving a labile repressor protein may be responsible for the inhibition of RNA splicing since treatment of SL12.4 cells with the protein synthesis inhibitor cycloheximide reversibly induces a rapid and dramatic accumulation of fully spliced TCR-beta transcripts in the cytoplasm, concomitant with a decline in TCR-beta pre-mRNAs in the nucleus. This inducible system may be useful for future studies analyzing the underlying molecular mechanisms that regulate RNA splicing.
Mol Cell Biol 1993 Mar
PMID:T cell receptor-beta mRNA splicing: regulation of unusual splicing intermediates. 844 6

The CI repressor protein from the temperate lambdoid phage HK022 was purified to near homogeneity and used in DNase I footprinting analyses to identify six binding sites in this phage. All these sites contained homologous 15 bp inverted repeats. Three of these 15 bp inverted repeats were located between the cI and cro (OR1 to OR3), and the other three were 3' to the cI gene (OL1 to OL3). Two of these sites were identified as operator sites for the repressor by DNA sequence analyses of virulent phage mutants. Almost all these mutations identified lay within the 15 bp inverted repeats comprising OR1 and OR2, and almost all were in the most highly conserved positions in the operators. The majority of virulent mutants contained mutations in both OR1 and OR2. Intrinsic affinities for individual operators were measured by DNase I footprinting analyses using DNA fragments which contained a single wild-type operator adjacent to two mutant operators. Comparison of these values with the affinity observed with these sites in the wild-type operator indicated that HK022 CI repressor bound cooperativity to OR1 and OR2 with a cooperativity parameter, omega, of almost 2000. Cooperative binding occurred in an alternative pairwise fashion, as previously seen with lambda CI repressor. In addition to cooperative binding between two adjacent operators, the repressor also increased the affinity for adjacent non-specific DNA sites, resulting in a periodic pattern of binding termed "phasing". This phasing pattern extended beyond regions predicted for pair-wise interaction, but was significantly decreased on a template with two adjacent operators, suggesting that pairwise cooperativity interfered with phasing.
J Mol Biol 1993 Apr 20
PMID:Highly cooperative DNA binding by the coliphage HK022 repressor. 848 97


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