DNA-bound transcription factor complexes analysed by mass-spectrometry: binding of novel proteins to the human c-fos SRE and related sequences

Transcription factors control eukaryotic polymerase II function by influencing the recruitment of multiprotein complexes to promoters and their subsequent integrated function. The complexity of the functional ‘transcriptosome’ has necessitated biochemical fractionation and subsequent protein sequencing on a grand scale to identify individual components. As a consequence, much is now known of the basal transcription complex. In contrast, less is known about the complexes formed at distal promoter elements. The c-fos SRE, for example, is known to bind Serum Response Factor (SRF) and ternary complex factors such as Elk-1. Their interaction with other factors at the SRE is implied but, to date, none have been identified. Here we describe the use of mass-spectrometric sequencing to identify six proteins, SRF, Elk-1 and four novel proteins, captured on SRE duplexes linked to magnetic beads. This approach is generally applicable to the characterisation of nucleic acid-bound protein complexes and the post-translational modification of their components.

Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity.

The cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1/Sdi1, important for p53-dependent cell cycle control, mediates G1/S arrest through inhibition of Cdks and possibly through inhibition of DNA replication. Cdk inhibition requires a sequence of approximately 60 amino acids within the p21 NH2 terminus. We show, using proteolytic mapping, circular dichroism spectropolarimetry, and nuclear magnetic resonance spectroscopy, that p21 and NH2-terminal fragments that are active as Cdk inhibitors lack stable secondary or tertiary structure in the free solution state. In sharp contrast to the disordered free state, however, the p21 NH2 terminus adopts an ordered stable conformation when bound to Cdk2, as shown directly by NMR spectroscopy. We have, thus, identified a striking disorder-order transition for p21 upon binding to one of its biological targets, Cdk2. This structural transition has profound implications in light of the ability of p21 to bind and inhibit a diverse family of cyclin-Cdk complexes, including cyclin A-Cdk2, cyclin E-Cdk2, and cyclin D-Cdk4. Our findings suggest that the flexibility, or disorder, of free p21 is associated with binding diversity and offer insights into the role for structural disorder in mediating binding specificity in biological systems. Further, these observations challenge the generally accepted view of proteins that stable secondary and tertiary structure are prerequisites for biological activity and suggest that a broader view of protein structure should be considered in the context of structure-activity relationships.

Magnetic charachterization of surface sediments in the South Atlantic

Surface sediment samples representative for the tropical and subtropical South Atlantic (15°N to 40°S) were investigated by isothermal magnetic methods to delineate magnetic mineral distribution patterns and to identify their predominant Holocene climatic and oceanographic controls. Individual parameters reveal distinct, yet frequently overlapping, regional sedimentation characteristics. A probabilistic ('fuzzy c-means') cluster analysis was applied to five concentration independent magnetic properties assessing magnetite to hematite ratios and diagnostic of bulk and fine-particle magnetite grain size and coercivity spectra. The resultant 10 cluster structures establish an oceanwide magnetic sediment classification scheme tracing the major terrigenous eolian and fluvial fluxes, authigenic biogenic magnetite accumulation in high-productivity areas, transport by ocean current systems, and effects of bottom water velocity on depositional regimes. Distinct dissimilarities in magnetic mineral inventories between the eastern and western basins of the South Atlantic reflect prominent contrasts of both oceanic and continental influences.

Petrography, chemistry and magnetic properties of ODP Leg 115 basalts

A detailed study of the Fe-Ti oxides in four basalt samples-one from each of the four holes drilled into basement on Ocean Drilling Program Leg 115 (Sites 706, 707, 713, and 715) has been performed. Ilmenite is present only in samples from Sites 706 and 715. In the sample from Site 715, Ti-magnetite intergrowths are characteristic of subaerial (?) high-temperature oxy-exsolution; Ti-magnetite in the other three samples has experienced pervasive low-temperature oxidation to Ti-maghemite, as evidenced by the double-humped, irreversible, saturation magnetization vs. temperature (Js/T) curves. The bulk susceptibility of these samples, which are similar in terms of major element chemistry, varies by a factor of ~20 and correlates semiquantitatively with the modal abundance of Fe-Ti spinel, as determined by image analysis with an electron microprobe. The variation in Fe-Ti oxide abundance correlates with average grain size: fine-grained samples contain less Fe-Ti oxide. This prompts the speculation that the crystallization rate may also influence Fe-Ti oxide abundance.

(Table 1) Grain size results, statistical parameters and magnetic susceptibility values of disturbed sediments from ODP holes 204-1249C and 204-1245B

Soupy and mousse-like fabrics are disturbance sedimentary features that result from the dissociation of gas hydrate, a process that releases water. During the core retrieval process, soupy and mousse-like fabrics are produced in the gas hydrate-bearing sediments due to changes in pressure and temperature conditions. Therefore, the identification of soupy and mousse-like fabrics can be used as a proxy for the presence of gas hydrate in addition to other evidence, such as pore water freshening or anomalously cool temperature. We present here grain-size results, mineralogical composition and magnetic susceptibility data of soupy and mousse-like samples from the southern Hydrate Ridge (Cascadia accretionary complex) acquired during Leg 204 of the Ocean Drilling Program. In order to study the relationship between sedimentary texture and the presence of gas hydrates, we have compared these results with the main textural and compositional data available from the same area. Most of the disturbed analyzed samples from the summit and the western flank of southern Hydrate Ridge show a mean grain size coarser than the average mean grain size of the hemipelagic samples from the same area. The depositional features of the sediments are not recognised due to disturbance. However, their granulometric statistical parameters and distribution curves, and magnetic susceptibility logs indicate that they correspond to a turbidite facies. These results suggest that gas hydrates in the southern Hydrate Ridge could form preferentially in coarser grain-size layers that could act as conduits feeding gas from below the BSR. Two samples from the uppermost metres near the seafloor at the summit of the southern Hydrate Ridge show a finer mean grain-size value than the average of hemipelagic samples. They were located where the highest amount of gas hydrates was detected, suggesting that in this area the availability of methane gas was high enough to generate gas hydrates, even within low-permeability layers. The mineralogical composition of the soupy and mousse-like sediments does not show any specific characteristic with respect to the other samples from the southern Hydrate Ridge.

Paleomagnetic and rock magnetic properties of ODP Leg 173 sites, west Iberia continental margin

We present detailed paleomagnetic and rock magnetic results of rock samples recovered during Leg 173. The Leg 173 cores display a multicomponent magnetization nature. Variations in magnetic properties correlate with changes in lithology that result from differences in the abundance and size of magnetic minerals. The combined investigation suggests that the magnetic properties of the "fresher" peridotite samples from Site 1070 are controlled mainly by titanomagnetite, with a strong Verwey transition in the vicinity of 110 K, and with field- and frequency-dependent susceptibility curves that resemble those of titanomagnetites. These results are in excellent agreement with thermomagnetic characteristics where titanomagnetites with Curie temperature ~580°C were identified from the "fresher" peridotites. In contrast to the magnetic properties observed from the "fresher" peridotites, the low-temperature curves for the "altered" peridotites did not show any Verwey transition. Thermomagnetic analysis using the high-temperature vibrating sample magnetometer also failed to show evidence for titanomagnetites. The remanent magnetization is carried by a thermally unstable mineral that breaks down at ~420°C, probably maghemite. The field- and frequency-dependent relationships are also directly opposite to those in the reversal zone, with no signs of titanomagnetite characteristics. Altogether, these rock magnetic data seem to be sensitive indicators of alteration and support the contention that maghemite is responsible for the magnetic signatures displayed in the altered peridotites of the upper section. The magnetic minerals of the basement rocks from Sites 1068, 1069, and 1070 are of variable particle size but fall within the pseudo-single-domain size range (0.2-14 µm). The average natural remanent magnetization (NRM) intensity of recovered serpenitinized peridotite is typically on the order of 20 mA/m for samples from Site 1068, but ~120 mA/m for samples from Site 1070. The much stronger magnetization intensity of Site 1070 is apparently in excellent agreement with the observed magnetic anomaly high. Nearly half of the NRM intensity remained after 400°C demagnetization, suggesting that the remanence can contribute significantly to the marine magnetic anomaly.

Rock magnetic records and geochemical methods of sediment cores off NW Africa

Two gravity cores retrieved off NW Africa at the border of arid and subtropical environments (GeoB 13602-1 and GeoB 13601-4) were analyzed to extract records of Late Quaternary climate change and sediment export. We apply End Member (EM) unmixing to 350 acquisition curves of isothermal remanent magnetization (IRM). Our approach enables to discriminate rock magnetic signatures of aeolian and fluvial material, to determine biomineralization and reductive diagenesis. Based on the occurrence of pedogenically formed magnetic minerals in the fluvial and aeolian EMs, we can infer that goethite formed in favor to hematite in more humid climate zones. The diagenetic EM dominates in the lower parts of the cores and within a thin near-surface layer probably representing the modern Fe**2+/Fe**3+ redox boundary. Up to 60% of the IRM signal is allocated to a biogenic EM underlining the importance of bacterial magnetite even in siliciclastic sediments. Magnetosomes are found well preserved over most of the record, indicating suboxic conditions. Temporal variations of the aeolian and fluvial EMs appear to faithfully reproduce and support trends of dry and humid conditions on the continent. The proportion of aeolian to fluvial material was dramatically higher during Heinrich Stadials, especially during Heinrich Stadial 1. Dust export from the Arabian-Asian corridor appears to vary contemporaneous to increased dust fluxes on the continental margin of NW Africa emphasizing that melt-water discharge in the North Atlantic had an enormous impact on atmospheric dynamics.