Evolutionary clues to DNA polymerase III beta clamp structural mechanisms

The prokaryotic DNA polymerase III beta homodimeric clamp links the replication complex to DNA during polynucleotide synthesis. This clamp is loaded onto DNA and unloaded by the clamp loader complex, the delta subunit of which by itself can bind to and open the clamp. beta Clamps from diverse bacteria were examined using contrast hierarchical alignment and interaction network (CHAIN) analysis, a statistical approach that categorizes and measures the evolutionary constraints imposed on protein sequences by natural selection. Some constraints are subtle inasmuch as they are unique to certain bacteria. Examination of corresponding molecular interactions within structures of the Escherichia coli beta dimeric and delta-beta complexes reveals that N320, Y323 and R176, which are subject to very strong constraints, form a substructure that may serve as a platform for leveraging and directing delta-induced conformational changes. N320 may play a prominent role, as it is strategically situated between this substructure and regions linked to delta binding and opening of beta's dimeric interface. R176 appears to act as a relay between the delta binding site and the clamp's central hole. Other residues subject to strong constraints are likewise associated with structurally important features. For example, two pairs of interacting residues, R269/E304 and K74/E300, form salt bridges at the dimeric interface, while the C-terminal residues M362, P363, M364 and R365 appear to play key roles in delta binding. Q149 and K198 appear to sense DNA within the clamp's central hole while other residues may relay this information to the delta binding site. Mutagenesis experiments designed to explore possible mechanisms are proposed

Measurement of Water Vapor Condensation on Apple Surfaces during Controlled Atmosphere Storage

Apples are stored at temperatures close to 0 °C and high relative humidity (up to 95%) under controlled atmosphere conditions. Under these conditions, the cyclic operation of the refrigeration machine and the associated temperature fluctuations can lead to localized undershoots of the dew point on fruit surfaces. The primary question for the present study was to prove that such condensation processes can be measured under practical conditions during apple storage. Using the example of a measuring point in the upper apple layer of a large bin in the supply air area, this evidence was provided. Using two independent measuring methods, a wetness sensor attached to the apple surface and determination of climatic conditions near the fruit, the phases of condensation, namely active condensation and evaporation, were measured over three weeks as a function of the operating time of the cooling system components (refrigeration machine, fans, defrosting regime). The system for measurement and continuous data acquisition in the case of an airtight CA-storage room is presented and the influence of the operation of the cooling system components in relation to condensation phenomena was evaluated. Depending on the set point specifications for ventilation and defrost control, condensed water was present on the apple surface between 33.4% and 100% of the duration of the varying cooling/re-warming cycles

AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes

Using a combination of computer methods for iterative database searches and multiple sequence alignment, we show that protein sequences related to the AAA family of ATPases are far more prevalent than reported previously. Among these are regulatory components of Lon and Clp proteases, proteins involved in DNA replication, recombination, and restriction (including subunits of the origin recognition complex, replication factor C proteins, MCM DNA-licensing factors and the bacterial DnaA, RuvB, and McrB proteins), prokaryotic NtrC-related transcription regulators, the Bacillus sporulation protein SpoVJ, Mg2+, and Co2+ chelatases, the Halobacterium GvpN gas vesicle synthesis protein, dynein motor proteins, TorsinA, and Rubisco activase. Alignment of these sequences, in light of the structures of the clamp loader delta' subunit of Escherichia coli DNA polymerase III and the hexamerization component of N-ethylmaleimide-sensitive fusion protein, provides structural and mechanistic insights into these proteins, collectively designated the AAA+ class. Whole-genome analysis indicates that this class is ancient and has undergone considerable functional divergence prior to the emergence of the major divisions of life. These proteins often perform chaperone-like functions that assist in the assembly, operation, or disassembly of protein complexes. The hexameric architecture often associated with this class can provide a hole through which DNA or RNA can be thread; this may be important for assembly or remodeling of DNA-protein complexes

Combustion of Shock-Dispersed Flake Aluminum - High-Speed Visualization

Charges of 0.5 g PETN were used to disperse 1 g of flake aluminum in a rectangular test chamber of 4 liter inner volume and inner dimensions of approximately 10 cm x 10 cm x 40 cm. The subsequent combustion of the flake aluminum with the ambient air in the chamber gave rise to a highly luminous flame. The evolution of the luminous region was studied by means of high-speed cinematography. The high-speed camera is responsive to a broad spectral range in the visible and near infra-red. For a number of tests this response range was narrowed down by means of a band-pass filter with a center wavelength of 488 nm and a half-width of 23 nm. The corresponding images were expected to have a stronger temperature dependence than images obtained without the filter, thus providing better capability to highlight hot-spots. Emission in the range of the pass-band of the filter can be due to continuous thermal radiation from hot Al and Al{sub 2}O{sub 3} particles or to molecular band emission from gaseous AlO. A time-resolving spectrometer was improvised to inspect this topic. The results suggest that AlO emission occurs, but that the continuous spectrum is the dominating effect in our experiments

Proteomic analysis of interchromatin granule clusters

A variety of proteins involved in gene expression have been localized within mammalian cell nuclei in a speckled distribution that predominantly corresponds to interchromatin granule clusters (IGCs). We have applied a mass spectrometry strategy to identify the protein composition of this nuclear organelle purified from mouse liver nuclei. Using this approach, we have identified 146 proteins, many of which had already been shown to be localized to IGCs, or their functions are common to other already identified IGC proteins. In addition, we identified 32 proteins for which only sequence information is available and thus these represent novel IGC protein candidates. We find that 54% of the identified IGC proteins have known functions in pre-mRNA splicing. In combination with proteins involved in other steps of pre-mRNA processing, 81% of the identified IGC proteins are associated with RNA metabolism. In addition, proteins involved in transcription, as well as several other cellular functions, have been identified in the IGC fraction. However, the predominance of pre-mRNA processing factors supports the proposed role of IGCs as assembly, modification, and/or storage sites for proteins involved in pre-mRNA processing

Bayesian Centroid Estimation for Motif Discovery

Biological sequences may contain patterns that are signal important biomolecular functions; a classical example is regulation of gene expression by transcription factors that bind to specific patterns in genomic promoter regions. In motif discovery we are given a set of sequences that share a common motif and aim to identify not only the motif composition, but also the binding sites in each sequence of the set. We present a Bayesian model that is an extended version of the model adopted by the Gibbs motif sampler, and propose a new centroid estimator that arises from a refined and meaningful loss function for binding site inference. We discuss the main advantages of centroid estimation for motif discovery, including computational convenience, and how its principled derivation offers further insights about the posterior distribution of binding site configurations. We also illustrate, using simulated and real datasets, that the centroid estimator can differ from the maximum a posteriori estimator.Comment: 24 pages, 9 figure

In the absence of ATPase activity, pre-RC formation is blocked prior to MCM2-7 hexamer dimerization

The origin recognition complex (ORC) of Saccharomyces cerevisiae binds origin DNA and cooperates with Cdc6 and Cdt1 to load the replicative helicase MCM2–7 onto DNA. Helicase loading involves two MCM2–7 hexamers that assemble into a double hexamer around double-stranded DNA. This reaction requires ORC and Cdc6 ATPase activity, but it is unknown how these proteins control MCM2–7 double hexamer formation. We demonstrate that mutations in Cdc6 sensor-2 and Walker A motifs, which are predicted to affect ATP binding, influence the ORC–Cdc6 interaction and MCM2–7 recruitment. In contrast, a Cdc6 sensor-1 mutant affects MCM2–7 loading and Cdt1 release, similar as a Cdc6 Walker B ATPase mutant. Moreover, we show that Orc1 ATP hydrolysis is not involved in helicase loading or in releasing ORC from loaded MCM2–7. To determine whether Cdc6 regulates MCM2–7 double hexamer formation, we analysed complex assembly. We discovered that inhibition of Cdc6 ATPase restricts MCM2–7 association with origin DNA to a single hexamer, while active Cdc6 ATPase promotes recruitment of two MCM2–7 hexamer to origin DNA. Our findings illustrate how conserved Cdc6 AAA+ motifs modulate MCM2–7 recruitment, show that ATPase activity is required for MCM2–7 hexamer dimerization and demonstrate that MCM2–7 hexamers are recruited to origins in a consecutive process

Phyloscan: locating transcription-regulating binding sites in mixed aligned and unaligned sequence data

The transcription of a gene from its DNA template into an mRNA molecule is the first, and most heavily regulated, step in gene expression. Especially in bacteria, regulation is typically achieved via the binding of a transcription factor (protein) or small RNA molecule to the chromosomal region upstream of a regulated gene. The protein or RNA molecule recognizes a short, approximately conserved sequence within a gene's promoter region and, by binding to it, either enhances or represses expression of the nearby gene. Since the sought-for motif (pattern) is short and accommodating to variation, computational approaches that scan for binding sites have trouble distinguishing functional sites from look-alikes. Many computational approaches are unable to find the majority of experimentally verified binding sites without also finding many false positives. Phyloscan overcomes this difficulty by exploiting two key features of functional binding sites: (i) these sites are typically more conserved evolutionarily than are non-functional DNA sequences; and (ii) these sites often occur two or more times in the promoter region of a regulated gene. The website is free and open to all users, and there is no login requirement. Address: (http://bayesweb.wadsworth.org/phyloscan/)

Dynein structure and power stroke

Dynein ATPases are microtubule motors that are critical to diverse processes such as vesicle transport and the beating of sperm tails; however, their mechanism of force generation is unknown. Each dynein comprises a head, from which a stalk and a stem emerge. Here we use electron microscopy and image processing to reveal new structural details of dynein c, an isoform from Chlamydomonas reinhardtii flagella, at the start and end of its power stroke. Both stem and stalk are flexible, and the stem connects to the head by means of a linker approximately 10 nm long that we propose lies across the head. With both ADP and vanadate bound, the stem and stalk emerge from the head 10 nm apart. However, without nucleotide they emerge much closer together owing to a change in linker orientation, and the coiled-coil stalk becomes stiffer. The net result is a shortening of the molecule coupled to an approximately 15-nm displacement of the tip of the stalk. These changes indicate a mechanism for the dynein power stroke