Zinc-Regulated DNA Binding of the Yeast Zap1 Zinc-Responsive Activator

The Zap1 transcription factor of Saccharomyces cerevisiae plays a central role in zinc homeostasis by controlling the expression of genes involved in zinc metabolism. Zap1 is active in zinc-limited cells and repressed in replete cells. At the transcriptional level, Zap1 controls its own expression via positive autoregulation. In addition, Zap1's two activation domains are regulated independently of each other by zinc binding directly to those regions and repressing activation function. In this report, we show that Zap1 DNA binding is also inhibited by zinc. DMS footprinting showed that Zap1 target gene promoter occupancy is regulated with or without transcriptional autoregulation. These results were confirmed using chromatin immunoprecipitation. Zinc regulation of DNA binding activity mapped to the DNA binding domain indicating other parts of Zap1 are unnecessary for this control. Overexpression of Zap1 overrode DNA binding regulation and resulted in constitutive promoter occupancy. Under these conditions of constitutive binding, both the zinc dose response of Zap1 activity and cellular zinc accumulation were altered suggesting the importance of DNA binding control to zinc homeostasis. Thus, our results indicated that zinc regulates Zap1 activity post-translationally via three independent mechanisms, all of which contribute to the overall zinc responsiveness of Zap1

Immobilization of Escherichia coli RNA Polymerase and Location of Binding Sites by Use of Chromatin Immunoprecipitation and Microarrays

The genome-wide location of RNA polymerase binding sites was determined in Escherichia coli using chromatin immunoprecipitation and microarrays (chIP-chip). Cross-linked chromatin was isolated in triplicate from rifampin-treated cells, and DNA bound to RNA polymerase was precipitated with an antibody specific for the β′ subunit. The DNA was amplified and hybridized to “tiled” oligonucleotide microarrays representing the whole genome at 25-bp resolution. A total of 1,139 binding sites were detected and evaluated by comparison to gene expression data from identical conditions and to 961 promoters previously identified by established methods. Of the detected binding sites, 418 were located within 1,000 bp of a known promoter, leaving 721 previously unknown RNA polymerase binding sites. Within 200 bp, we were able to detect 51% (189/368) of the known σ70-specific promoters occurring upstream of an expressed open reading frame and 74% (273/368) within 1,000 bp. Conversely, many known promoters were not detected by chIP-chip, leading to an estimated 26% negative-detection rate. Most of the detected binding sites could be associated with expressed transcription units, but 299 binding sites occurred near inactive transcription units. This map of RNA polymerase binding sites represents a foundation for studies of transcription factors in E. coli and an important evaluation of the chIP-chip technique

Structure-Based Cyclic Glycoprotein Ibα-Derived Peptides Interfering with von Willebrand Factor-Binding, Affecting Platelet Aggregation under Shear

The plasmatic von Willebrand factor (VWF) circulates in a compact form unable to bind platelets. Upon shear stress, the VWF A1 domain is exposed, allowing VWF-binding to platelet glycoprotein Ib-V-IX (GPIbα chain). For a better understanding of the role of this interaction in cardiovascular disease, molecules are needed to specifically interfere with the opened VWF A1 domain interaction with GPIbα. Therefore, we in silico designed and chemically synthetized stable cyclic peptides interfering with the platelet-binding of the VWF A1 domain per se or complexed with botrocetin. Selected peptides (26–34 amino acids) with the lowest-binding free energy were: the monocyclic mono- vOn Willebrand factoR-GPIbα InTerference (ORbIT) peptide and bicyclic bi-ORbIT peptide. Interference of the peptides in the binding of VWF to GPIb-V-IX interaction was retained by flow cytometry in comparison with the blocking of anti-VWF A1 domain antibody CLB-RAg35. In collagen and VWF-dependent whole-blood thrombus formation at a high shear rate, CLB-RAg35 suppressed stable platelet adhesion as well as the formation of multilayered thrombi. Both peptides phenotypically mimicked these changes, although they were less potent than CLB-RAg35. The second-round generation of an improved peptide, namely opt-mono-ORbIT (28 amino acids), showed an increased inhibitory activity under flow. Accordingly, our structure-based design of peptides resulted in physiologically effective peptide-based inhibitors, even for convoluted complexes such as GPIbα-VWF A1

Distributed Energy Systems in California's Future: A Preliminary Report Volume 2

The construction and use of energy technologies produce environmental and social consequences that are neither desired nor, for the most part, incorporated in the economic costs charged for the energy supplied. Although it is now essentially universally recognized that these 'externalities' or (broadly defined) 'social costs' must somehow be taken into account in the processes by which society chooses among alternative energy options, it is less widely appreciated that these costs - not resource limits or narrow economics - actually define the energy dilemma in the long term. It is important to try to make clear at the outset why this is so. The energy problem resides fundamentally in the fact that the relation between energy and well-being is two-sided. The application of energy as a productive input to the economy, yielding desired goods and services, contributes to well-being; the environmental and social costs of getting and using energy subtract from it. At some level of energy use, and for a given mix of technologies of energy supply, further increases in energy supply will produce incremental social and environmental costs greater than the incremental economic benefits - that is, growth begins to do more harm than good (Holdren, 1977; Committee on Nuclear and Alternative Energy Systems, 1977). This level can be said to define a rational 'limit to growth', as distinct from a strictly physical one. That such a level, beyond which energy growth no longer pays, exists in principle for any mix of technologies of supply and end-use is easily shown from basic economics and physical science; predicting its magnitude exactly is much harder, the more so because social costs even less quantifiable than environmental ones may dominate. Lovins (1976, 1977) evidently believes that the United States is already near or beyond the point, given the 'hard' energy technologies on which it relies, where further growth hurts more than it helps. Whether he is right or wrong about exactly where we are now, however, or in specific judgments about the merits of 'hard' versus 'soft' technologies, it is clear that energy policy for the long term should be shaped by awareness that social-environmental costs, not exhaustion of resources, will limit the amount of human well-being derivable from energy. Maximizing this quantity will require striving for technologies of energy supply with low social and environmental costs per unit of energy delivered, and fostering patterns and technologies of energy end-use that squeeze from each such unit the maximum contribution to human well-being. This perspective, then, elevates environmental and social characteristics to the top of the list of criteria used to select supply technologies from the menu of genuinely long-term options - fission breeder reactors, fusion, direct and indirect harnessing of solar flows, and possibly some forms of geothermal energy. It rationalizes the possibility that society will choose to pay more (in economic terms) for a more benign energy source than for a less benign one. And it argues for using, as a criterion for selecting short-term and transition energy sources, the extent to which these promote and facilitate the transition to a longer term energy future built on more benign sources and efficient end-use. Given a perspective that places environmental and social impacts at the heart of the energy predicament rather than on the periphery, it becomes essential to compare the impacts produced by alternative energy options systematically, comprehensively, and objectively. The information needed to do this properly, even for a limited set of technologies and a limited geographic and cultural context (e.g., California), unfortunately does not exist. What is attempted here, therefore, is to outline a logical framework for such a comparison, and to hang on that framework the partial information that is available on the environmental impacts of some major conventional and nonconventional energy options for California. Although the emphasis in this study is on the latter, the most sensible yardstick to give meaning to the results is provided by the former. The objective is to permit at least some partial and preliminary conclusions about this aspect of the 'soft' energy options, and to identify those areas where additional knowledge is most badly needed. In this analysis sociopolitical impacts are mentioned from time to time for completeness, but the emphasis is on impacts on physical resources and on the physical environment; impacts on institutions and social systems per se are treated more thoroughly in other papers in this project