The effects of DNA cytosine methylation on H -ras promoter structure and function

The effect of DNA cytosine methylation on H-ras promoter activity was assessed using a transient expression system employing the plasmid H-rasCAT (NaeI H-ras promoter linked to the chloramphenicol acetyltransferase (CAT) gene). This 551 bp promoter is 80% GC rich, enriched with 168 CpG dinucleotides, and contains six functional GC box elements which represent major DNA methylation target sites. Prokaryotic methyltransferases HhaI (CGm$\sp5$CG) and HpaII (Cm$\sp5$CGG) alone or in combination with a human placental methyltransferase (HP MTase) were used to introduce methyl groups at different CpG sites within the promoter. To test for functional promoter activity, the methylated plasmids were introduced into CV-1 cells and CAT activity assessed 48 h post-transfection. Methylation at specific HhaI and HpaII sites reduced CAT expression by 70%, whereas more extensive methylation at generalized CpG sites with HP MTase inactivated the promoter $>$95%. The inhibition of H-ras promoter activity was not attributable to methylation-induced differences in DNA uptake or stability in the cell, topological form of the plasmid, or methylation effects in nonpromoter regions. We also observed that DNA cytosine methylation of a 360 bp promoter fragment by HP MTase induced a local change in DNA conformation. Using three independent methodologies (nitrocellulose filter binding assays, gel mobility shifts, and Southwestern blots), we determined that this change in promoter conformation affected the interaction of nuclear proteins with cis-regulatory sequences residing in the promoter region. The results provide evidence to suggest that DNA methylation may regulate gene expression by inducing changes in local promoter conformation which in turn alters the interactions between DNA and protein factors required for transcription. The results provide supportive evidence for the hypothesis of Cedar and Riggs, who postulated that DNA methylation may regulate gene expression by altering the binding affinities of proteins for DNA. ^

Electronic structure of copper nitrides as a function of nitrogen content

he nitrogen content dependence of the electronic properties for copper nitride thin films with an atomic percentage of nitrogen ranging from 26 ± 2 to 33 ± 2 have been studied by means of optical (spectroscopic ellipsometry), thermoelectric (Seebeck), and electrical resistivity measurements. The optical spectra are consistent with direct optical transitions corresponding to the stoichiometric semiconductor Cu3N plus a free-carrier contribution, essentially independent of temperature, which can be tuned in accordance with the N-excess. Deviation of the N content from stoichiometry drives to significant decreases from − 5 to − 50 μV/K in the Seebeck coefficient and to large enhancements, from 10− 3 up to 10 Ω cm, in the electrical resistivity. Band structure and density of states calculations have been carried out on the basis of the density functional theory to account for the experimental results.

Structure of intermediate shocks in collisionsless anisotropic Hall-magnetohydrodynamics plasma models

The existence of discontinuities within the double-adiabatic Hall-magnetohydrodynamics (MHD) model is discussed. These solutions are transitional layers where some of the plasma properties change from one equilibrium state to another. Under the assumption of traveling wave solutions with velocity C and propagation angle θ with respect to the ambient magnetic field, the Hall-MHD model reduces to a dynamical system and the waves are heteroclinic orbits joining two different fixed points. The analysis of the fixed points rules out the existence of rotational discontinuities. Simple considerations about the Hamiltonian nature of the system show that, unlike dissipative models, the intermediate shock waves are organized in branches in parameter space, i.e., they occur if a given relationship between θ and C is satisfied. Electron-polarized (ion-polarized) shock waves exhibit, in addition to a reversal of the magnetic field component tangential to the shock front, a maximum (minimum) of the magnetic field amplitude. The jumps of the magnetic field and the relative specific volume between the downstream and the upstream states as a function of the plasma properties are presented. The organization in parameter space of localized structures including in the model the influence of finite Larmor radius is discussed

Structure and function of the Bacillus hybrid enzyme GluXyn-1: Native-like jellyroll fold preserved after insertion of autonomous globular domain

The 1,3–1,4-β-glucanase from Bacillus macerans (wtGLU) and the 1,4-β-xylanase from Bacillus subtilis (wtXYN) are both single-domain jellyroll proteins catalyzing similar enzymatic reactions. In the fusion protein GluXyn-1, the two proteins are joined by insertion of the entire XYN domain into a surface loop of cpMAC-57, a circularly permuted variant of wtGLU. GluXyn-1 was generated by protein engineering methods, produced in Escherichia coli and shown to fold spontaneously and have both enzymatic activities at wild-type level. The crystal structure of GluXyn-1 was determined at 2.1 Å resolution and refined to R = 17.7% and R(free) = 22.4%. It shows nearly ideal, native-like folding of both protein domains and a small, but significant hinge bending between the domains. The active sites are independent and accessible explaining the observed enzymatic activity. Because in GluXyn-1 the complete XYN domain is inserted into the compact folding unit of GLU, the wild-type-like activity and tertiary structure of the latter proves that the folding process of GLU does not depend on intramolecular interactions that are short-ranged in the sequence. Insertion fusions of the GluXyn-1 type may prove to be an easy route toward more stable bifunctional proteins in which the two parts are more closely associated than in linear end-to-end protein fusions.

A cobalt complex that selectively disrupts the structure and function of zinc fingers

Zinc finger domains are structures that mediate sequence recognition for a large number of DNA-binding proteins. These domains consist of sequences of amino acids containing cysteine and histidine residues tetrahedrally coordinated to a zinc ion. In this report, we present a means to selectively inhibit a zinc finger transcription factor with cobalt(III) Schiff-base complexes. 1H NMR spectroscopy confirmed that the structure of a zinc finger peptide is disrupted by axial ligation of the cobalt(III) complex to the nitrogen of the imidazole ring of a histidine residue. Fluorescence studies reveal that the zinc ion is displaced from the model zinc finger peptide in the presence of the cobalt complex. In addition, gel-shift and filter-binding assays reveal that cobalt complexes inhibit binding of a complete zinc finger protein, human transcription factor Sp1, to its consensus sequence. Finally, a DNA-coupled conjugate of the cobalt complexes selectively inhibited Sp1 in the presence of several other transcription factors.

Microbes as engines of ecosystem function : When does community structure enhance predictions of ecosystem processes?

Peer reviewed

Structure and function in rhodopsin: Packing of the helices in the transmembrane domain and folding to a tertiary structure in the intradiscal domain are coupled*

A previous study of the retinitis pigmentosa mutation L125R and two designed mutations at this site, L125A and L125F, showed that these mutations cause partial or total misfolding of the opsins expressed in COS cells from the corresponding mutant opsin genes. We now report on expression and characterization of the opsins from the following retinitis pigmentosa mutants in the transmembrane domain of rhodopsin that correspond to six of the seven helices: G51A and G51V (helix A), G89D (helix B), A164V (helix D), H211P (helix E), P267L and P267R (helix F), and T297R (helix G). All the mutations caused partial misfolding of the opsins as observed by the UV/visible absorption characteristics and by separation of the expressed opsins into fractions that bound 11-cis-retinal to form the corresponding mutant rhodopsins and those that did not bind 11-cis-retinal. Further, all the mutant rhodopsins prepared from the above mutants, except for G51A, showed strikingly abnormal bleaching behavior with abnormal metarhodopsin II photointermediates. The results show that retinitis pigmentosa mutations in every one of the transmembrane helices can cause misfolding of the opsin. Therefore, on the basis of these and previous results, we conclude that defects in the packing of the transmembrane helices resulting from these mutations are relayed to the intradiscal domain, where they cause misfolding of the opsin by inducing the formation of a disulfide bond other than the native Cys-110—Cys-187 disulfide bond. Thus, there is coupling between packing of the helices in the transmembrane domain and folding to a tertiary structure in the intradiscal domain.

Structure and function in rhodopsin: Topology of the C-terminal polypeptide chain in relation to the cytoplasmic loops*

Cysteine mutagenesis and site-directed spin labeling in the C-terminal region of rhodopsin have been used to probe the local structure and proximity of that region to the cytoplasmic loops. Each of the native amino acids in the sequence T335–T340 was replaced with Cys, one at a time. The sulfhydryl groups of all mutants reacted rapidly with the sulfhydryl reagent 4,4′-dithiodipyridine, which indicated a high degree of solvent accessibility. Furthermore, to probe the proximity relationships, a series of double Cys mutants was constructed. One Cys in all sets was at position 338 and the other was at a position in the sequence S240–V250 in the EF interhelical loop, at position 65 in the AB interhelical loop, or at position 140 in the CD interhelical loop. In the dark state, no significant disulfide formation was observed between C338 and C65 or C140 under the conditions used, whereas a relatively rapid disulfide formation was observed between C338 and C242 or C245. Spin labels in the double Cys mutants showed the strongest magnetic interactions between the nitroxides attached to C338 and C245 or C246. Light activation of the double mutant T242C/S338C resulted in slower disulfide formation, whereas interactions between nitroxides at C338 and C245 or C246 decreased. These results suggest the proximity of the C-terminal residue C338 to residues located on the outer face of a cytoplasmic helical extension of the F helix with an apparent increase of distance upon photoactivation.

Structure and function in rhodopsin: Rhodopsin mutants with a neutral amino acid at E134 have a partially activated conformation in the dark state*

The Glu-134–Arg-135 residues in rhodopsin, located near the cytoplasmic end of the C helix, are involved in G protein binding, or activation, or both. Furthermore, the charge-neutralizing mutation Glu-134 to Gln-134 produces hyperactivity in the activated state and produces constitutive activity in opsin. The Glu/Asp-Arg charge pair is highly conserved in equivalent positions in other G protein-coupled receptors. To investigate the structural consequences of charge-neutralizing mutations at Glu-134 and Arg-135 in rhodopsin, single spin-labeled side chains were introduced at sites in the cytoplasmic domains of helices C (140), E (227), F (250), or G (316) to serve as “molecular sensors” of the local helix bundle conformation. In each of the spin-labeled rhodopsins, a Gln substitution was introduced at either Glu-134 or Arg-135, and the electron paramagnetic resonance spectrum of the spin label was used to monitor the structural response of the helix bundle. The results indicate that a Gln substitution at Glu-134 induces a photoactivated conformation around helices C and G even in the dark state, an observation of potential relevance to the hyperactivity and constitutive activity of the mutant. In contrast, little change is induced in helix F, which has been shown to undergo a dominant motion upon photoactivation. This result implies that the multiple helix motions accompanying photoactivation are not strongly coupled and can be induced to take place independently. Gln substitution at Arg-135 produces only minor structural changes in the dark- or light-activated conformation, suggesting that this residue is not a determinant of structure in the regions investigated, although it may be functionally important.