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Al–SiC and hybrid Al–SiC-graphite MMCs with up to 70 vol% SiC and 10 vol% graphite were fabricated using spark plasma sintering. Detailed studies of the microstructure, Vickers hardness, longitudinal elastic constant C11, as well as thermal shock resistance of the MMC samples were carried out. Non-destructive ultrasound phase spectroscopy was used to measure the C11 elastic constant of the MMCs, and the measured values were compared with different micromechanical models for particle reinforced composites. For both two- and three-component MMCs, the Hashin Shtrikman lower bound fitted best to the experimental results in the absence of any porosity. Thermal shock resistance of the MMCs was carried out by heating in air to 500 °C and subsequently quenching in water for ten times. The extent of thermal shock-induced structural alteration was determined via a systematic study of the change in density, hardness, and C11 of the samples. It is observed that the thermal shock resistance of the MMCs depends upon multiple factors such as SiC content, graphite content, and the amount of residual porosity. While the reduction in mechanical properties of the Al–SiC MMCs due to graphite addition is according to the expected line, the present study shows that graphite addition also reduces the thermal shock resistance of the MMC.

Metal/ceramic composites are in high demand in several industries because of their superior thermo-mechanical properties. Among various composite types, the interpenetrating phase composites (IPCs) with percolating metallic and ceramic phases offer manifold benefits, such as a good combination of strength, toughness, and stiffness, very good thermal properties, excellent wear resistance, as well as the flexibility of microstructure and processing route selection, etc. The fabrication of metal/ceramic IPCs typically involves two steps - i) processing of an open porous ceramic body, and ii) infiltration of metallic melt in the pores to fabricate the IPC. Although significant progress has been made in recent years for developing both porous ceramics and melt infiltration methods, to the best of the knowledge of the authors, no review article summarizing all the aspects of processing and properties of IPCs has been published till date. This review article is aimed at filling this gap. Starting with a brief introduction about the current status and applications of IPCs, the various processing routes for fabricating open porous ceramic preforms and melt infiltration techniques have been discussed. Subsequently, the data available for various important physical, mechanical, and thermal properties for IPCs have been critically analyzed to thoroughly understand their dependence on various structural and processing parameters. To compare the properties of IPCs with other relevant materials, seven different Ashby material property maps have been used, and the domains for IPCs have been created in them. For each map, the concept of material indices has been employed to critically discuss how IPCs perform in relation to other material classes for various optimum design conditions. Finally, a detailed future outlook for further research on IPCs has been provided.

Interpenetrating phase composites (IPCs) are a class of composites consisting of two or more topologically co-continuous phases and have three-dimensionally percolating interconnected structure. The co-continuous and interconnected architecture of the IPCs can provide superior properties compared to the conventional particulate reinforced composites at room and elevated temperatures. IPCs having metal/ceramic phases are finding applications in various fields like (thermal management materials, aerospace, automobile industries, light-weight armor materials, etc.). In the present work, Al-Si alloy/SiC foam IPC was fabricated by infiltrating the alloy melt into SiC foam via the squeeze-casting process. Open porous SiC foams, fabricated via the replication method, and having two different pore sizes (10 and 20 PPI) were used for IPC fabrication. The SiC foams had both macropores between the SiC foam struts and micropores within the strut walls. Results show that the Brinell hardness of the composite fabricated using a small pore size (20 PPI) SiC foam is higher than the composite fabricated with a large pore size SiC foam (10 PPI). Thorough structural characterization via scanning electron microscope and localized study of hardness distribution in different regions of the composite using nanoindentation were carried out.

Objectives Individuals with limited health literacy often experience suboptimal health outcomes. This study examined the frequency of limited health literacy and demographic and psychosocial factors associated with limited health literacy in a sample of older Black Americans. Methods Participants (n = 330) enrolled in a community-based intervention to promote colorectal cancer (CRC) screening completed baseline surveys assessing health literacy with the Rapid Estimate of Adult Literacy in Medicine, Revised (REALM-R) test, CRC awareness, cancer fatalism, Preventive Health Model (PHM) constructs, and demographics. Results Approximately 52% of participants had limited health literacy, the REALM-R score was 5.4 (SD = 2.7). Univariable correlates of limited health literacy were gender, employment, income, prior screening, cancer fatalism, CRC awareness, and PHM constructs (religious beliefs, salience/coherence, perceived susceptibility). Multivariable correlates of limited health literacy were male gender (OR = 2.3, CI = 1.4–3.8), unable to work (OR = 2.8, CI = 1.3–6.1), lower household income (OR = 3.0, CI = 1.6, 5.5), and higher PHM religious beliefs (OR = 1.1, CI = 1.0–1.2). Conclusion Limited health literacy was associated with multiple complex factors. Interventions should incorporate patient health literacy and low-literacy materials that can be delivered through multiple channels. Practice implications Future studies are needed to understand the role of health literacy in an individual’s health behavior and the provision of effective healthcare.

Several architectures namely Cloud, Fog, Mist, and Edge computing have been proposed for IoT systems, but efforts to practically correlate between them have proved hollow due to difficulties in creating live systems that replicate the frameworks. In this paper, a comparison of these architectures has been made. Simulation models have been developed to form an understanding of the variation in their performance for different values of configuration and workload parameters in a parallel multi-query processing environment. To derive realistic simulations for sensor-data, the Citybench smart city dataset has been used, which is extracted from measurements taken by traffic, temperature, and other sensors, along with a wide range of queries generated in a smart city application. By using seven highly contrasting single and six ratio based mixed Queries, it can be seen that Edge with proximity to query devices and sensors achieves the least latency in four of the single and five of the mixed query simulations with Mist being a close second in the same. However, Fog due to its distributed and high computational capability obtains minimum latency for two single query simulations with Cloud being best for compute-intensive cases.

Routine biosolids monitoring data provides an independent and comprehensive means to estimate water lead release pre-, during and post-Flint Water Crisis (FWC). The mass of potable plumbing-related metals ( i.e., lead, cadmium, copper, nickel and zinc) in sewage biosolids strongly correlated with one another during the FWC (p < 0.05). A simple parametric regression model based on 90th percentile potable water lead measurements (WLL90) from five city-wide citizen science sampling efforts August 2015-August 2017 was strongly correlated to corresponding monthly lead mass in biosolids [Biosolids-Pb (kg) = 0.483 x WLL90 (mu g/L) + 1.79: R-2 = 0.86, p < 0.05]. Although total biosolids lead increased just 14% during the 18 months of the FWC versus the comparable time pre-FWC, 76% of that increase occurred in July -September 2014, and the corresponding percentage of Flint children under 6 years with elevated blood lead >= 5 mu g/dL (i.e., %EBL5) doubling from 3.45% to 6.61% in those same three months versus 2013 (p < 0.05). %EBL5 was not statistically higher during the remaining months of the FWC compared to preFWC or post-FWC. As expected, lead in biosolids during the FWC, when orthophosphate was not added, was moderately correlated with water temperature (R-2 = 0.30, p < 0.05), but not at other times pre- and post-FWC when orthophosphate was present. Tripling the orthophosphate dose post-FWC versus pre-FWC and some lead pipe removal, decreased lead in biosolids (and %EBLS) to historic lows (2016-2017 vs. 2012-2013; p < 0.05), supporting the effectiveness of these public health interventions in reducing childhood water lead exposure. (C) 2019 The Authors. Published by Elsevier Ltd. U.S. Environmental Protection Agency [8399375] This publication was partly funded and developed under Grant No. 8399375 "Untapping the Crowd: Consumer Detection and Control of Lead in Drinking Water" awarded by the U.S. Environmental Protection Agency to Virginia Tech. It has not been formally reviewed by EPA. The views expressed in this document are solely those of the authors and do not necessarily reflect those of the Agency. EPA does not endorse any products or commercial services mentioned in this publication.

The mechanism of internal load transfer in a newly developed interpenetrating metal/ceramic composite is studied for the first time in this work using energy dispersive synchrotron X-ray diffraction. The composite was fabricated by infiltration of Al12Si melt in an open porous alumina preform, fabricated using a mixture of two different polymer waxes as pore formers, by gas pressure infiltration. Several diffraction planes of all three crystalline phases of the composite – alumina, silicon and aluminum solid solution are investigated. Lattice strains, both along and transverse to the loading direction, in each phase are calculated from the shifts in the measured diffraction peak positions. Results show that until about 100 MPa applied compressive stress, all three phases undergo elastic deformation. At higher applied stresses aluminum undergoes plastic deformation and correspondingly load is transferred to alumina. The load carrying capability of alumina is however limited by incipient damage in this phase. At applied compressive stresses higher than 187 MPa, load carried by the alumina phase continuously decreases and correspondingly more load is again carried by the aluminum solid solution. Throughout, the fraction of load carried by silicon remains almost constant, suggesting no significant load transfer within the metallic alloy itself.

DNA and core histones are hierarchically packaged into a complex organization called chromatin. The nucleosome assembly protein (NAP) family of histone chaperones is involved in the deposition of histone complexes H2A/H2B and H3/H4 onto DNA and prevents nonspecific aggregation of histones. Testis-specific Y-encoded protein (TSPY)-like protein 5 (TSPYL5) is a member of the TSPY-like protein family, which has been previously reported to interact with ubiquitin-specific protease USP7 and regulate cell proliferation and is thus implicated in various cancers, but its interaction with chromatin has not been investigated. In this study, we characterized the chromatin association of TSPYL5 and found that it preferentially binds histone H3/H4 via its C-terminal NAP-like domain both in vitro and ex vivo. We identified the critical residues involved in the TSPYL5-H3/H4 interaction and further quantified the binding affinity of TSPYL5 toward H3/H4 using biolayer interferometry. We then determined the binding stoichiometry of the TSPYL5-H3/H4 complex in vitro using a chemical cross-linking assay and size-exclusion chromatography coupled with multiangle laser light scattering. Our results indicate that a TSPYL5 dimer binds to either two histone H3/H4 dimers or a single tetramer. We further demonstrated that TSPYL5 has a specific affinity toward longer DNA fragments and that the same histone-binding residues are also critically involved in its DNA binding. Finally, employing histone deposition and supercoiling assays, we confirmed that TSPYL5 is a histone chaperone responsible for histone H3/H4 deposition and nucleosome assembly. We conclude that TSPYL5 is likely a new member of the NAP histone chaperone family.

Rising incidence of waterborne diseases including Legionellosis linked to low chlorine residuals in buildings and the availability of inexpensive testing options, create an opportunity for citizen science chorine monitoring to complement sampling done by water utilities. University researchers and Flint residents coordinated a citizen science chlorine surveillance campaign in Flint, Michigan in 2015-19, that helped expose the nature of two deadly Legionnaires Disease outbreaks in 2014-2015 during the Flint Water Crisis and progress of system recovery during the Federal emergency. Results obtained with an inexpensive color wheel were in agreement with a digital colorimeter (R2 =0.99; p = 2.81 × 10-21) at 15 sites geographically distributed across Flint. Blinded tests revealed good agreement between official (n = 2051) and citizen (n = 654) data in terms of determining whether regulatory guidelines for chlorine were met, but a discovery that the citizen data were statistically lower than the city's (p

A systematic methodology for developing hybrid Al–SiC-graphite functionally graded composite materials with the highest feasible SiC-content in the top-most layer, without resulting in residual porosities, has been established. The distribution of ceramic reinforcements in individual layers is very homogeneous, and the interlayer regions are free from any defect. Monolithic composites, having composition identical to the overall average compositions of the FGMs, were also fabricated, and their mechanical properties were compared with the corresponding FGM properties. Due to the locally high SiC-content, the top-layer hardness of the FGM is significantly higher than the corresponding monolithic composite. A systematic study of the flexural stress-strain behavior of individual mono-layers and the overall FGM was carried out at different orientations. For identical mono-layer compositions, the orientation of the FGM has a strong influence on its flexural stress-strain behavior. The failure stress is significantly higher when the layer containing high SiC-content is at the compressively loaded side during the 4-point bend test.

Thermal expansion behavior of an interpenetrating metal/ceramic composite having ceramic contents in the range of 34−60 vol.% is studied in this work. Four thermal cycles were carried out between room temperature and 500 °C at a constant heating/cooling rate of 5 °C/min. Both thermal strain and thermal expansion coefficient decrease with increasing ceramic content in the composite. Evolution of the thermal expansion coefficient with temperature shows anisotropic nature. At all ceramic contents, highest thermal strain and thermal expansion coefficient are obtained along the preform press direction. The extent of thermal expansion anisotropy depends upon both temperature and ceramic content in the composite. The evolution of the observed thermal expansion anisotropy has been attributed to the elastic anisotropy of the preforms and composites, considering as well the elastic-plastic flow behavior of the metallic matrix. The results have been compared with several analytical theoretical models.

The elastic–plastic deformation and internal load transfer in metal/ceramic composites are studied in this work both numerically and experimentally. The composite was fabricated by squeeze-casting AlSi12 melt in an open porous preform made by freeze-casting and drying of alumina suspension. Such composites exhibit a complex microstructure composed of lamellar domains. Single-domain samples were extracted from bulk material. Uniaxial compression tests were carried out parallel to the direction of the alternating metallic alloy and ceramic lamellae in the plane normal to the direction of freeze-casting. This loading mode is selected as highest load transfer occurs when loaded along the ceramic lamellae. Numerical modeling was done using the finite element method using quasi-3D microstructure based on metallographic 2D section and a modified Voigt homogenization technique assuming plastic behavior of the metallic alloy, absence of any damage and ideal interface between the phases. Internal load transfer mechanism was predicted for composites with different ceramic volume fractions. Results show that at any applied stress, as the ceramic content increases, the phase stress in alumina along the loading direction continuously decreases. Experimental validation of the numerical results is carried out by in-situ compression test along with energy dispersive synchrotron X-ray diffraction in one sample with 41 vol% ceramic. Results show that both the numerical techniques yield similar results, which match well with the experimental measurements. The ratio of the phase stress to the applied stress in alumina reaches a highest value between 2 and 2.5 up to a compressive stress of about 300 MPa. At higher applied stresses both the experimentally determined lattice microstrain and the phase stress along the loading direction in alumina decrease due to the initiation of possible damage. This study shows that the applied economic and more flexible homogenization technique is a viable tool for modeling of this composite structure.

The internal load transfer and compressive damage evolution in an interpenetrating Al 2 O 3 /AlSi12 composite have been studied in this work. The composite was fabricated by squeeze-casting eutectic aluminium–silicon alloy melt in a porous alumina preform. The preform was fabricated from a mixture of cellulose fibres and alumina particles via cold pressing and sintering. In an earlier work we reported the internal load transfer in the same composite material under monotonic compression and tension studied using energy dispersive synchrotron X-ray diffraction [20] . The current work is a continuation of this earlier study, aimed at obtaining further understanding about load transfer occurring during load reversal and damage behaviour during external compression. The micromechanical load partitioning between the three phases present in the composite is studied during one load cycle starting in compression followed by unloading and reloading in tension until failure. Average strain and stress value in each phase is calculated from several diffraction planes of each phase and as a result the reported strain and stress are representative of the bulk material behaviour. The load transfer results allow identifying the occurrence of a substantial Bauschinger effect in the Al solid solution phase and progressive damage evolution within the alumina phase. In situ compression test inside a scanning electron microscope showed that failure of the composite occurred by propagation of cracks through the ceramic rich regions, oriented at approximately 45° to the loading direction.

The complete stiffness matrices of several metal/ceramic composites were analysed using the complementary ultrasonic spectroscopic techniques ultrasound phase spectroscopy (UPS) and resonant ultrasound spectroscopy (RUS). Three different aluminum/alumina composites having complex interpenetrating architectures were studied: a composite based on freeze-cast ceramic preform, a composite based on open porous ceramic preform obtained by pyrolysis of cellulose fibres, and a composite based on discontinuous fibre preform. Six of the nine independent elastic constants describing orthotropic elastic anisotropy were pre-determined by ultrasound phase spectroscopy and used as initial guess input for resonant ultrasound spectroscopy analysis, making the final results of all nine elastic constants more reliable. In all cases, consistent and reproducible results are obtained. Finally the experimental results were compared with effective elastic constants calculated using micromechanical modelling and a good correspondence between both is obtained.

Four-phosphate-adaptor protein 1 (FAPP1) regulates secretory transport from the trans-Golgi network (TGN) to the plasma membrane. FAPP1 is recruited to the Golgi through binding of its pleckstrin homology (PH) domain to phosphatidylinositol 4-phosphate (PtdIns(4)P) and a small GTPase ADP-ribosylation factor 1 (ARF1). Despite the critical role of FAPP1 in membrane trafficking, the molecular basis of its dual function remains unclear. Here, we report a 1.9 Å resolution crystal structure of the FAPP1 PH domain and detail the molecular mechanisms of the PtdIns(4)P and ARF1 recognition. The FAPP1 PH domain folds into a seven-stranded β-barrel capped by an α-helix at one edge, whereas the opposite edge is flanked by three loops and the β4 and β7 strands that form a lipid-binding pocket within the β-barrel. The ARF1-binding site is located on the outer side of the β-barrel as determined by NMR resonance perturbation analysis, mutagenesis, and measurements of binding affinities. The two binding sites have little overlap, allowing FAPP1 PH to associate with both ligands simultaneously and independently. Binding to PtdIns(4)P is enhanced in an acidic environment and is required for membrane penetration and tubulation activity of FAPP1, whereas the GTP-bound conformation of the GTPase is necessary for the interaction with ARF1. Together, these findings provide structural and biochemical insight into the multivalent membrane anchoring by the PH domain that may augment affinity and selectivity of FAPP1 toward the TGN membranes enriched in both PtdIns(4)P and GTP-bound ARF1.

An interpenetrating composite fabricated by squeeze-casting AlSi12 melt in a porous alumina preform is studied in this work. The preform was fabricated by pyrolysis of cellulose fibres used as a pore-forming agent and subsequent sintering of alumina particles. The resulting preform had both micropores within the ceramic walls and macropores between those walls, which were infiltrated by the liquid metal. Lattice strains under externally applied tension and compression in all three phases of the composite were measured using energy dispersive synchrotron X-ray diffraction. Multiple diffraction peaks in each phase were analysed, allowing information about the interplanar anisotropy of each phase to be obtained. Results show that load transfer occurs from aluminium to stiffer and stronger alumina and silicon phases in both elastic and plastic regions. Stresses as well as stress concentration factors along the loading direction in all three phases were calculated from the measured continuum mechanics equivalent lattice strain and the macroscopic elastic constants of each phase. The stress concentration factor in aluminium is less than unity throughout, and it decreases with increasing applied stress owing to load transfer. The maximum stress in alumina along the loading direction is reached in the region of plastic deformation of aluminium. Subsequently, at applied stresses higher than −300 MPa in the sample under compression, stress in alumina along the loading direction decreases owing to damage.

The tumor suppressor p53 and the DNA repair factor 53BP1 (p53 binding protein 1) regulate gene transcription and responses to genotoxic stresses. Upon DNA damage, p53 undergoes dimethylation at Lys382 (p53K382me2), and this posttranslational modification is recognized by 53BP1. The molecular mechanism of nonhistone methyl-lysine mark recognition remains unknown. Here we report a 1. 6-A-resolution crystal structure of the tandem Tudor domain of human 53BP1 bound to a p53K382me2 peptide. In the complex, dimethylated Lys382 is restrained by a set of hydrophobic and cation–π interactions in a cage formed by four aromatic residues and an aspartate of 53BP1. The signature HKKme2 motif of p53, which defines specificity, is identified through a combination of NMR resonance perturbations, mutagenesis, measurements of binding affinities and docking simulations, and analysis of the crystal structures of 53BP1 bound to p53 peptides containing other dimethyl-lysine marks, p53K370me2 (p53 dimethylated at Lys370) and p53K372me2 (p53 dimethylated at Lys372). Binding of the 53BP1 Tudor domain to p53K382me2 may facilitate p53 accumulation at DNA damage sites and promote DNA repair as suggested by chromatin immunoprecipitation and DNA repair assays. Together, our data detail the molecular mechanism of p53–53BP1 association and provide the basis for deciphering the role of this interaction in the regulation of p53 and 53BP1 functions.

Aluminium/alumina composites based on ceramic preforms prepared via freeze-casting are examined. Domains composed of alternating but also interpenetrating ceramic and metallic lamellae are observed. Single-domain samples were extracted from composites processed under different conditions. In situ scanning electron microscopy analyses were carried out to investigate the damage evolution under compressive load. The composite is strong and brittle when loaded along directions parallel to the freezing direction. When compressed in other directions, the behavior is controlled by the soft metal. The plastic anisotropy is less pronounced than theoretical predictions for laminates, which is explained by the presence of bridges between the ceramic lamellae. Coating the preform with Cu or Cu2O prior to melt infiltration reduces the compressive strengths of the composites. Transmission electron microscopy analysis shows that even in the case of a Cu coating a Cu2O layer is formed during processing, weakening the interface and preventing the dissolution of Cu in the aluminium alloy.

The elastic properties of single domains of lamellar AlSi12/Al2O3 composites produced by metal infiltration of freeze cast preforms have been examined. The anisotropic elastic constants determined from ultrasonic phase spectroscopy (UPS) experiments have been compared to microstructure based FE-models created with the program OOF2, micromechanical models (Mori–Tanaka and inverse Mori–Tanaka) and an analytical model for an ideal laminate. The influences of lamellae orientations and ceramic contents on the elastic constants have been investigated. Along the lamellae directions the microstructure based FE model and the inverse Mori–Tanaka model are in good agreement with experimental results. Perpendicular to the lamellae the experiment shows a stiffer than expected elastic behaviour.

Innovative metal/ceramic composites produced by melt infiltration of ceramic preforms prepared by a freeze-casting technique are examined in this study. These composites exhibit a characteristic hierarchical structure: On a mesoscopic length scale, lamellar domains with sizes up to several millimetres are observed. The aim of the present study was to analyze the anisotropic elastic properties of such a composite on different length scales. Experimental studies were carried out on samples containing about 44% alumina ceramic and 56% aluminium-based alloy (Al–12Si). Ultrasonic wave velocity measurements were carried out on macroscopic poly-domain samples as well as on miniature single-domain samples. The elastic constants derived from these measurements are discussed in light of elasticity models established for lamellar and for fibrous composites.

A second DNA binding protein from stationary-phase cells of Mycobacterium smegmatis (MsDps2) has been identified from the bacterial genome. It was cloned, expressed and characterised and its crystal structure was determined. The core dodecameric structure of MsDps2 is the same as that of the Dps from the organism described earlier (MsDps1). However, MsDps2 possesses a long N-terminal tail instead of the C-terminal tail in MsDps1. This tail appears to be involved in DNA binding. It is also intimately involved in stabilizing the dodecamer. Partly on account of this factor, MsDps2 assembles straightway into the dodecamer, while MsDps1 does so on incubation after going through an intermediate trimeric stage. The ferroxidation centre is similar in the two proteins, while the pores leading to it exhibit some difference. The mode of sequestration of DNA in the crystalline array of molecules, as evidenced by the crystal structures, appears to be different in MsDps1 and MsDps2, highlighting the variability in the mode of Dps-DNA complexation. A sequence search led to the identification of 300 Dps molecules in bacteria with known genome sequences. Fifty bacteria contain two or more types of Dps molecules each, while 195 contain only one type. Some bacteria, notably some pathogenic ones, do not contain Dps. A sequence signature for Dps could also be derived from the analysis.

The structure of the DNA binding protein from starved cells from Mycobacterium smegmatis has been determined in three crystal forms and has been compared with those of similar proteins from other sources. The dodecameric molecule can be described as a distorted icosahedron. The interfaces among subunits are such that the dodecameric molecule appears to have been made up of stable trimers. The situation is similar in the proteins from Escherichia coli and Agrobacterium tumefaciens, which are closer to the M. smegmatis protein in sequence and structure than those from other sources, which appear to form a dimer first. Trimerisation is aided in the three proteins by the additional N-terminal stretches that they possess. The M. smegmatis protein has an additional C-terminal stretch compared to other related proteins. The stretch, known to be involved in DNA binding, is situated on the surface of the molecule. A comparison of the available structures permits a delineation of the rigid and flexible regions in the molecule. The subunit interfaces around the molecular dyads, where the ferroxidation centres are located, are relatively rigid. Regions in the vicinity of the acidic holes centred around molecular 3-fold axes, are relatively flexible. So are the DNA binding regions. The crystal structures of the protein from M. smegmatis confirm that DNA molecules can occupy spaces within the crystal without disturbing the arrangement of the protein molecules. However, contrary to earlier suggestions, the spaces do not need to be between layers of protein molecules. The cubic form provides an arrangement in which grooves, which could hold DNA molecules, criss-cross the crystal.

The cyclic AMP receptor protein (CRP) acts as a transcription activator at many promoters of Escherichia coli. We have examined the kinetics of open complex formation at the lacP1 promoter using tryptophan fluorescence of RNA polymerase and DNA fragments with 2-aminopurine substituted at specific positions. Apart from the closed complex formation and promoter clearance, we were able to detect three steps. The first step after the closed complex formation leads to a rapid increase of 2-aminopurine fluorescence. This was followed by another rapid step in which quenching of tryptophan fluorescence of RNA polymerase was observed. The slowest step detected by 2-aminopurine fluorescence increase is assigned to the final open complex formation. We have found that CRP not only enhances RNA polymerase binding at the promoter, but also enhances the slowest isomerization step by about 2-fold. Furthermore, potassium permanganate probing shows that the conformation of the open complex in the presence of CRP appears qualitatively and quantitatively different from that in the absence of CRP, suggesting that contact with RNA polymerase is maintained throughout the transcription initiation.

Activation and repression of transcription are primarily caused by gene regulatory proteins (activators and repressors), which act by binding to specific sites on DNA. The steps from initial binding of RNA polymerase to the elongating complex are characterized by many intermediates, each with a discrete structure, offering many mechanistic possibilities for regulator actions. It has been shown in some systems that the activator acts by helping RNA polymerase or other associated factors to bind (recruitment) and/or by influencing a postrecruitment step (isomerization, promoter clearance, etc.) (1-7). We have used the term recruitment for referring to assistance only on the initial binding step of RNA polymerase. We caution that a postbinding step may be indistinguishable from the recruitment step if they are in rapid equilibrium. Clearly, all activators do not act at the level of RNA polymerase recruitment to the promoters. There are activators demonstrated to help postbinding steps that have no effect on initial binding (4-7). Promoter-specific repression can occur by sterically hindering the binding of RNA polymerase or of, in principle, another essential transcription factor to the promoter (8, 9). However, other studies in several promoters, as was anticipated (10), point toward repressor action also through contact with promoter-bound RNA polymerase at a postbinding step (11-17). More interestingly, some regulators act as activator in one context and as repressor in another (13, 15). Although the contact regions on the surface of some regulators and of RNA polymerase have been mapped (18, 19), how these contacts cause activation or inhibition of transcription initiation in biochemical terms is not known. In principle, the contact may affect the process of transcription initiation (i) by allosteric modification of RNA polymerase and/or (ii) by energetic stabilization of an intermediate(s). Regulator-induced conformation changes in RNA polymerase by protein-protein contact may contribute to the regulation process. However, a regulator-RNA polymerase contact may play a fundamentally different role in transcription initiation. In this article, we provide a conceptual framework for the process of activator and repressor action through differential stabilization of one or more of the intermediate states of RNA polymerase-promoter complex by its contact with the regulator. We portray regulators as catalysts. From a thermodynamic point, we view that activators, like catalysts, lower the activation energy of some step(s) in the reaction pathway of transcription initiation. As discussed below, a similar energetic argument explains the action of repressors. To make our point, we discuss simple examples of DNA-binding regulators modulating RNA polymerase during transcription initiation in selected prokaryotic systems.

Only cytosine contains two adjacent aromatic protons that give rise to cross-peaks in the aromatic region of 2D-COSY spectra of oligodeoxynucleotides. In two GC-containing sequences several such cross-peaks were resolved. The intensity of these cross-peaks is a sensitive monitor of local mobility, and upon the addition of the intercalating drug daunomycin selective intensity losses were observed, indicating binding to specific GC base pairs. We have also monitored the 2D-COSY cross-peaks from mobile pyrimidine bases in tRNA (Phe) as a function of temperature.