Lathyrism: has the scenario changed in 2013?

Lathyrism is now rarely seen as a clinical disease in general, medical or neurology outpatient departments, throughout the world. Eating patterns of seeds of Lathyrus sativus are still prevalent focal points in parts of the world. Question arises, why are we not seeing cases of lathyrism? Is it that the disease has changed its profile, with the changing socioeconomic status of the poor or underdeveloped or moderately developed countries? Is it that the seeds of lathyrus are less toxic now? Is it that the body defence against toxins of lathyrus has genetically modified? To find out answers to these interesting questions, an extensive questionnaire-based sampling was done among 1000 subjects from northern India to identify the human behaviour regarding the knowledge, attitude, and practices (KAPs) for L. sativus. Four clinically suspected cases of Lathyrism were also fully worked up. It was concluded that many areas of India are still being fed with lathyrus seeds, but not many cases have appeared. Many questions have to be answered, as to what has reduced the incidence of lathyrism.

Towards a minimal motif for artificial transcriptional activators.

BACKGROUND: Most transcriptional activators minimally comprise two functional modules, one for DNA binding and the other for activation. Several activators also bear an oligomerization region and bind DNA as dimers or higher order oligomers. In a previous study we substituted these domains of a protein activator with synthetic counterparts [Mapp et al., Proc. Natl. Acad. Sci. USA 97 (2000) 3930-3935]. An artificial transcriptional activator, 4.2 kDa in size, comprised of a DNA binding hairpin polyamide tethered to a 20 residue activating peptide (AH) was shown to stimulate promoter specific transcription [Mapp et al., Proc. Natl. Acad. Sci. USA 97 (2000) 3930-3935]. The question arises as to the general nature and the versatility of this minimal activator motif and whether smaller ligands can be designed which maintain potent activation function. RESULTS: Here we have replaced the 20 amino acid AH peptide with eight or 16 residues derived from the activation domain of the potent viral activator VP16. The 16 residue activation module coupled to the polyamide activated transcription over two-fold better than the analogous AH conjugate. Altering the site of attachment of the activation module on the polyamide allowed reduction of the intervening linker from 36 atoms to eight without significant diminution of the activation potential. In this study we also exchanged the polyamide to target a different sequence without compromising the activation function further demonstrating the generality of this design. CONCLUSIONS: The polyamide activator conjugates described here represent a class of DNA binding ligands which are tethered to a second functional moiety, viz. an activation domain, that recruits elements of the endogenous transcriptional machinery. Our results define the minimal structural elements required to construct artificial, small molecule activators. If such activators are cell-permeable and can be targeted to designated sites in the genome, this series of conjugates may then serve as a tool to study mechanistic aspects of transcriptional regulation and eventually to modulate gene expression relevant to human diseases.

Recruitment of the transcriptional machinery through GAL11P: structure and interactions of the GAL4 dimerization domain.

The GAL4 dimerization domain (GAL4-dd) is a powerful transcriptional activator when tethered to DNA in a cell bearing a mutant of the GAL11 protein, named GAL11P. GAL11P (like GAL11) is a component of the RNA-polymerase II holoenzyme. Nuclear magnetic resonance (NMR) studies of GAL4-dd revealed an elongated dimer structure with C(2) symmetry containing three helices that mediate dimerization via coiled-coil contacts. The two loops between the three coiled coils form mobile bulges causing a variation of twist angles between the helix pairs. Chemical shift perturbation analysis mapped the GAL11P-binding site to the C-terminal helix alpha3 and the loop between alpha1 and alpha2. One GAL11P monomer binds to one GAL4-dd dimer rendering the dimer asymmetric and implying an extreme negative cooperativity mechanism. Alanine-scanning mutagenesis of GAL4-dd showed that the NMR-derived GAL11P-binding face is crucial for the novel transcriptional activating function of the GAL4-dd on GAL11P interaction. The binding of GAL4 to GAL11P, although an artificial interaction, represents a unique structural motif for an activating region capable of binding to a single target to effect gene expression.

Interaction of a transcriptional repressor with the RNA polymerase II holoenzyme plays a crucial role in repression.

The yeast transcriptional repressor Tup1, tethered to DNA, represses to strikingly different degrees transcription elicited by members of two classes of activators. Repression in both cases is virtually eliminated by mutation of either member of the cyclin-kinase pair Srb10/11. In contrast, telomeric chromatin affects both classes of activators equally, and in neither case is that repression affected by mutation of Srb10/11. In vitro, Tup1 interacts with RNA polymerase II holoenzyme bearing Srb10 as well as with the separated Srb10. These and other findings indicate that at least one aspect of Tup1's action involves interaction with the RNA polymerase II holoenzyme.

Activation of gene expression by small molecule transcription factors.

Eukaryotic transcriptional activators are minimally comprised of a DNA binding domain and a separable activation domain; most activator proteins also bear a dimerization module. We have replaced these protein modules with synthetic counterparts to create artificial transcription factors. One of these, at 4.2 kDa, mediates high levels of DNA site-specific transcriptional activation in vitro. This molecule contains a sequence-specific DNA binding polyamide in place of the typical DNA binding region and a nonprotein linker in place of the usual dimerization peptide. Thus our activating region, a designed peptide, functions outside of the archetypal protein context, as long as it is tethered to DNA. Because synthetic polyamides can, in principle, be designed to recognize any specific sequence, these results represent a key step toward the design of small molecules that can up-regulate any specified gene.

An artificial transcriptional activating region with unusual properties.

We describe a series of transcriptional activators generated by adding amino acids (eight in one case, six in another) to fragments of the yeast Saccharomyces cerevisiae activator Gal4 that dimerize and bind DNA. One of the novel activating regions identified by this procedure is unusual, compared with previously characterized yeast activating regions, in the following ways: it works more strongly than does Gal4's natural activating region as assayed in yeast; it is devoid of acidic residues; and several lines of evidence suggest that it sees targets in the yeast transcriptional machinery at least partially distinct from those seen by Gal4's activating region.

A transcriptional activating region with two contrasting modes of protein interaction.

A C-terminal segment of the yeast activator Gal4 manifests two functions: When tethered to DNA, it elicits gene activation, and it binds the inhibitor Gal80. Here we examine the effects on these two functions of cysteine and proline substitutions. We find that, although certain cysteine substitutions diminish interaction with Gal80, those substitutions have little effect on the activating function in vivo and interaction with TATA box-binding protein (TBP) in vitro. Proline substitutions introduced near residues critical for Gal80 binding abolish that interaction but once again have no effect on the activating function. Crosslinking experiments show that a defined position in the activating peptide is in close proximity to TBP and Gal80 in the two separate reactions and show that binding of the inhibitor blocks binding to TBP. Thus, the same stretch of amino acids are involved in two quite different protein-protein interactions: binding to Gal80, which depends on a precise sequence and the formation of a defined secondary structure, or interactions with the transcriptional machinery in vivo, which are not impaired by perturbations of either sequence or structure.

An activator target in the RNA polymerase II holoenzyme.

Expression of protein-coding genes in eukaryotes involves the recruitment, by transcriptional activator proteins, of a transcription initiation apparatus consisting of greater than 50 polypeptides. Recent genetic and biochemical evidence in yeast suggests that a subset of these proteins, called SRB proteins, are likely targets for transcriptional activators. We demonstrate here, through affinity chromatography, photo-cross-linking, and surface plasmon resonance experiments, that the GAL4 activator interacts directly with the SRB4 subunit of the RNA polymerase II holoenzyme. The GAL4 activation domain binds to two essential segments of SRB4. The physiological relevance of this interaction is confirmed by mutations in SRB4, which occur within its GAL4-binding domain and which restore activation in vivo by a GAL4 derivative bearing a mutant activation domain.

DNA-bend modulation in a repressor-to-activator switching mechanism.

Recent discoveries of activator proteins that distort DNA but bear no obvious activation domains have focused attention on the role of DNA structure in transcriptional regulation. Here we describe how the transcription factor MerR can mediate repression as well as activation through stereospecific modulation of DNA structure. The repressor form of MerR binds between the -10 and -35 promoter elements of the bacterial mercury-detoxification genes, PT, allowing RNA polymerase to form an inactive complex with PT and MerR at this stress-inducible promoter. Upon mercuric ion binding, Hg-MerR converts this polymerase complex into the transcriptionally active or 'open' form. We show here that MerR bends DNA towards itself in a manner similar to the bacterial catabolite-activator protein CAP, namely at two loci demarked by DNase I sensitivity, and that the activator conformation, Hg-MeR, relaxes these bends. This activator-induced unbending, when coupled with the previously described untwisting of the operator, remodels the promoter and makes it a better template for the poised polymerase.

Construction and characterization of a mercury-independent MerR activator (MerRAC): transcriptional activation in the absence of Hg(II) is accompanied by DNA distortion.

The MeR regulatory protein of transposon Tn501 controls the expression of the mercury resistance (mer) genes in response to the concentration of mercuric ions. MerR is unique among prokaryotic regulatory proteins so far described in that it acts as a repressor [-Hg(II)] and an activator [+Hg(II)] of transcription of the mer genes, but binds to a single site on the DNA in both cases. This transcriptional activation process has been postulated to involve a protein-induced conformational change in the DNA that allows RNA polymerase more readily to form an open complex at the promoter. It has been shown [Frantz and O'Halloran (1990) Biochemistry, 29, 4747-4751] that activation of transcription by MerR in the presence of mercury is accompanied by hypersensitivity of the operator to chemical nucleases that are sensitive to local distortion in DNA structure. Here we describe specific mutations in MerR that allow the protein to stimulate transcription in the absence of the allosteric activator Hg(II). We demonstrate that the degree of activation caused by these mutants directly correlates with the degree of DNA distortion as measured by the hypersensitivity of MerR-DNA complexes to the nuclease Cu-5-phenyl-o-phenanthroline. These results support the model described above.

Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR.

Positive control of transcription often involves stimulatory protein-protein interactions between regulatory factors and RNA polymerase. Critical steps in the activation process itself are seldom ascribed to protein-DNA distortions. Activator-induced DNA bending is typically assigned a role in binding-site recognition, alterations in DNA loop structures or optimal positioning of the activator for interaction with polymerase. Here we present a transcriptional activation mechanism that does not require a signal-induced DNA bend but rather a receptor-induced untwisting of duplex DNA. The allosterically modulated transcription factor MerR is a repressor and an Hg(II)-responsive activator of bacterial mercury-resistance genes. Escherichia coli RNA polymerase binds to the MerR-promoter complex but cannot proceed to a transcriptionally active open complex until Hg(II) binds to MerR (ref. 6). Chemical nuclease studies show that the activator form, but not the repressor, induces a unique alteration of the helical structure localized at the centre of the DNA-binding site. Data presented here indicate that this Hg-MerR-induced DNA distortion corresponds to a local underwinding of the spacer region of the promoter by about 33 degrees relative to the MerR-operator complex. The magnitude and the direction of the Hg-MerR-induced change in twist angle are consistent with a positive control mechanism involving reorientation of conserved, but suboptimally phased, promoter elements and are consistent with a role for torsional stress in formation of an open complex.