Biomedical subjects

Patrick Schultz

Publications and source records attributed to Patrick Schultz.

13 recordsLinked to original sources

PubMed2026

Systematic common and rare variant association testing in 392,030 whole genomes in All of Us.

Large-scale genome-wide association studies (GWAS) and rare variant association studies (RVAS) from population biobanks provide valuable resources for gene discovery in complex human traits. We present an analysis of the All of Us Research Program v8 release, which includes whole genome sequencing data and harmonized phenotypic information of 392,030 participants after quality control, enabling a unified investigation of rare and common variants across a spectrum of human traits and diseases. We build an extensive phenome- and genome-wide ("All by All") computational framework to perform GWAS and RVAS on 3,602 phenotypes and identify 49,863 approximately independent, high-quality single-variant and gene-level associations. Meta-analyses of All of Us and UK Biobank, with sample sizes as large as 786,871 participants, further enhance statistical power and find 193 pLoF gene-phenotype associations that are not significant in either cohort alone, including 22 associations not highlighted by previous studies. We also present a public interactive browser that integrates association results for common and rare variants to facilitate interpretation and rapid querying of summary statistics, along with supporting documentation, and a Featured Workspace in the All of Us Researcher Workbench. Our framework will apply to iterative data releases as All of Us grows, empowering researchers worldwide to uncover insights into the functional effects of genetic components on complex traits and diseases.

Journal Article↗

PubMed2006

Formation of well-defined soluble aggregates upon fusion to MBP is a generic property of E6 proteins from various human papillomavirus species.

Protein aggregation is a main barrier hindering structural and functional studies of a number of interesting biological targets. The E6 oncoprotein of Human Papillomavirus strain 16 (E6(16)) is difficult to express under a native soluble form in bacteria. Produced as an unfused sequence, it forms inclusion bodies. Fused to the C-terminus of MBP, it is mainly produced in the form of soluble high molecular weight aggregates. Here, we produced as MBP-fusions seven E6 proteins from other HPV strains (5, 11, 18, 33, 45, 52, and 58) belonging to four different species, and we compared their aggregation state to that of MBP-E6(16). Using a fast mutagenesis method, we changed most non-conserved cysteines to the isosteric residue serine to minimize disulfide bridge-mediated aggregation during purification. Static and dynamic light scattering measurements, ultracentrifugation and electron microscopy demonstrated the presence in all MBP-E6 preparations of soluble high-molecular weight aggregates with a well-defined spherical shape. These aggregated particles are relatively monodisperse but their amount and their size vary depending on the conditions of expression and the strain considered. For all strains, minimal aggregate formation occurs when the expression is performed at 15 degrees C. Such observations suggest that the assembly of MBP-E6 aggregates takes place in vivo during protein biosynthesis, rather than occurring during purification. Finally, we show that all MBP-E6 preparations contain two zinc ions per protein monomer, suggesting that E6 domains within the high molecular weight aggregates possess a native-like fold, which enables correct coordination to the metal center.

Alphapapillomavirus↗

PubMed2006

Structure and oligomeric state of human transcription factor TFIIE.

The general RNA polymerase II transcription factor TFIIE, which is composed of two subunits, has essential roles in both transcription initiation and promoter escape. Electron microscopy analysis of negatively stained human TFIIE showed a large proportion of alpha/beta heterodimers as well as a small proportion of tetramers. Analytical ultracentrifugation, chemical crosslinking, pulldown experiments and cryo-electron microscopy confirmed that TFIIE is a alpha/beta heterodimer in solution. Three-dimensional envelopes of the alpha/beta particles showed an elongated structure composed of three distinct modules. Finally, a model for the quaternary architecture of the complex is proposed that provides a structural framework to discuss the function of TFIIE in the context of RNA polymerase II transcription initiation.

Dimerization↗

PubMed2004

Molecular architecture of the S. cerevisiae SAGA complex.

The Saccharomyces cerevisiae SAGA complex is a multifunctional coactivator that regulates transcription by RNA polymerase II. The 3D structure of SAGA, revealed by electron microscopy, is formed by five modular domains and shows a high degree of structural conservation to human TFTC, reflecting their related subunit composition. The positions of several SAGA subunits were mapped by immunolabeling and by analysis of mutant complexes. The Taf (TBP-associated factor) subunits, shared with TFIID, occupy a central region in SAGA and form a similar structure in both complexes. The locations of two histone fold-containing core subunits, Spt7 and Ada1, are consistent with their role in providing a SAGA-specific interface with the Tafs. Three components that perform distinct regulatory functions, Spt3, Gcn5, and Tra1, are spatially separated, underscoring the modular nature of the complex. Our data provide insights into the molecular architecture of SAGA and imply a functional organization to the complex.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Molecular architecture of the basal transcription factor B-TFIID.

BTAF1 (formerly named TAF(II)170/TAF-172) is an essential, evolutionarily conserved member of the SNF2-like family of ATPase proteins and together with TATA-binding protein (TBP) forms the B-TFIID complex. BTAF1 has been proposed to play a key role in the dynamic regulation of TBP function in RNA polymerase II transcription. We have determined the structure of native B-TFIID purified from human cells by electron microscopy and by image analysis of single particles at a resolution of 28 A. B-TFIID is 15 x 9 nm in size and is organized into a large domain of about 170 kDa, which can be subdivided into two domains. Extending from this domain is a long thumb, which in turn is divided into subdomains of about 25, 15, and 35 kDa, the largest of which is located at the end of the thumb. Immunolabeling experiments localize the extreme carboxyl terminus of BTAF1 within the 170-kDa domain, whereas the amino terminus and TBP co-localize to the end of the protruding thumb. The central portion of BTAF1 localizes to the base of the thumb. Comparison of the native B-TFIID with its recombinant form shows that both share a similar domain organization. Collectively, these data provide the first structural model of the B-TFIID complex and map its key functional domains.

Adenosine Triphosphate↗

PubMed2004

Mapping key functional sites within yeast TFIID.

The transcription factor TFIID, composed of the TATA box-binding protein (TBP) and 14 TBP-associated factors (TAFs), plays a key role in the regulation of gene expression by RNA polymerase II. The structure of yeast TFIID, as determined by electron microscopy and digital image analysis, is formed by three lobes, labelled A-C, connected by thin linking domains. Immunomapping revealed that TFIID contains two copies of the WD-40 repeat-containing TAF5 and that TAF5 contributes to the linkers since its C- and N-termini were found in different lobes. This property was confirmed by the finding that a recombinant complex containing TAF5 complexed with six histone fold containing TAFs was able to form a trilobed structure. Moreover, the N-terminal domain of TAF1 was mapped in lobe C, whereas the histone acetyltransferase domain resides in lobe A along with TAF7. TBP was found in the linker domain between lobes A and C in a way that the N-terminal 100 residues of TAF1 are spanned over it. The implications of these data with regard to TFIID function are discussed.

Acetyltransferases↗

PubMed2004

Immobilization of biotinylated DNA on 2-D streptavidin crystals.

The structural study of transient nucleoprotein complexes by electron microscopy is hampered by the coexistence of multiple interaction states leading to an heterogeneous image population. To tackle this problem, we have investigated the controlled immobilization of double stranded DNA molecules and of nucleoprotein complexes onto a support suitable for cryo-electron microscopy observation. The DNA was end-labeled with a biotin moiety in order to decorate, or to be incorporated into, two-dimensional streptavidin crystals formed in contact of a biotinylated lipid layer. The binding specificity and efficiency were examined by radioactively labeled oligonucleotides and by direct visualization of unstained and hydrated nucleic acid molecules in cryo-electron microscopy. By using RNA polymerase we further show that, once immobilized, femtomolar amounts of DNA template are suitable to interact with the enzyme. The image analysis of the RNA polymerase-DNA complexes showed that a three-dimensional model can be retrieved from such samples.

Biotinylation↗

PubMed2003

Cryo-negative staining reveals conformational flexibility within yeast RNA polymerase I.

The structure of the yeast DNA-dependent RNA polymerase I (RNA Pol I), prepared by cryo-negative staining, was studied by electron microscopy. A structural model of the enzyme at a resolution of 1.8 nm was determined from the analysis of isolated molecules and showed an excellent fit with the atomic structure of the RNA Pol II Delta4/7. The high signal-to-noise ratio (SNR) of the stained molecular images revealed a conformational flexibility within the image data set that could be recovered in three-dimensions after implementation of a novel strategy to sort the "open" and "closed" conformations in our heterogeneous data set. This conformational change mapped in the "wall/flap" domain of the second largest subunit (beta-like) and allows a better accessibility of the DNA-binding groove. This displacement of the wall/flap domain could play an important role in the transition between initiation and elongation state of the enzyme. Moreover, a protrusion was apparent in the cryo-negatively stained model, which was absent in the atomic structure and was not detected in previous 3D models of RNA Pol I. This structure could, however, be detected in unstained views of the enzyme obtained from frozen hydrated 2D crystals, indicating that this novel feature is not induced by the staining process. Unexpectedly, negatively charged molybdenum compounds were found to accumulate within the DNA-binding groove, which is best explained by the highly positive electrostatic potential of this region of the molecule, thus, suggesting that the stain distribution reflects the overall surface charge of the molecule.

Binding Sites↗

PubMed2003

Supramolecular self-assembly of lipid derivatives on carbon nanotubes.

Images of the assembly of surfactants and synthetic lipids on the surface of carbon nanotubes were obtained by transmission electron microscopy. Above the critical micellar concentration, sodium dodecyl sulfate (SDS) forms supramolecular structures made of rolled-up half-cylinders on the nanotube surface. Depending on the symmetry and the diameter of the carbon nanotube, we observed rings, helices, or double helices. Similar self-assemblies were also obtained with several synthetic single-chain lipids designed for the immobilization of histidine-tagged proteins. At the nanotube-water interface, permanent assemblies were produced from mixed micelles of SDS and different water-insoluble double-chain lipids after dialysis of the surfactant. Such arrangements could be further exploited for the development of new biosensors and bioelectronic nanomaterials.

Adsorption↗

PubMed2002

The A14-A43 heterodimer subunit in yeast RNA pol I and their relationship to Rpb4-Rpb7 pol II subunits.

A43, an essential subunit of yeast RNA polymerase I (pol I), interacts with Rrn3, a class I general transcription factor required for rDNA transcription. The pol I-Rrn3 complex is the only form of enzyme competent for promoter-dependent transcription initiation. In this paper, using biochemical and genetic approaches, we demonstrate that the A43 polypeptide forms a stable heterodimer with the A14 pol I subunit and interacts with the common ABC23 subunit, the yeast counterpart of the omega subunit of bacterial RNA polymerase. We show by immunoelectronic microscopy that A43, ABC23, and A14 colocalize in the three-dimensional structure of the pol I, and we demonstrate that the presence of A43 is required for the stabilization of both A14 and ABC23 within the pol I. Because the N-terminal half of A43 is clearly related to the pol II Rpb7 subunit, we propose that the A43-A14 pair is likely the pol I counterpart of the Rpb7-Rpb4 heterodimer, although A14 distinguishes from Rpb4 by specific sequence and structure features. This hypothesis, combined with our structural data, suggests a new localization of Rpb7-Rpb4 subunits in the three-dimensional structure of yeast pol II.

Amino Acid Sequence↗

PubMed2002

Localization of the yeast RNA polymerase I-specific subunits.

The spatial distribution of four subunits specifically associated to the yeast DNA-dependent RNA polymerase I (RNA pol I) was studied by electron microscopy. A structural model of the native enzyme was determined by cryo-electron microscopy from isolated molecules and was compared with the atomic structure of RNA pol II Delta 4/7, which lacks the specific polypeptides. The two models were aligned and a difference map revealed four additional protein densities present in RNA pol I, which were characterized by immunolabelling. A protruding protein density named stalk was found to contain the RNA pol I-specific subunits A43 and A14. The docking with the atomic structure showed that the stalk protruded from the structure at the same site as the C-terminal domain (CTD) of the largest subunit of RNA pol II. Subunit A49 was placed on top of the clamp whereas subunit A34.5 bound at the entrance of the DNA binding cleft, where it could contact the downstream DNA. The location of the RNA pol I-specific subunits is correlated with their biological activity.

Cryoelectron Microscopy↗

PubMed2002

Mapping histone fold TAFs within yeast TFIID.

The transcription factor TFIID is a large multiprotein complex, composed of the TATA box-binding protein (TBP) and 14 TBP-associated factors (TAFs), which plays a key role in the regulation of gene expression by RNA polymerase II. The three-dimensional structure of yeast (y) TFIID, determined at approximately 3 nm resolution by electron microscopy and image analysis, resembles a molecular clamp formed by three major lobes connected by thin linking domains. The yTFIID is structurally similar to the human factor although the clamp appears more closed in the yeast complex, probably reflecting the conformational flexibility of the structure. Immunolabelling experiments showed that nine TAFs that contain the histone fold structural motif were located in three distinct substructures of TFIID. The distribution of these TAFs showed that the previously reported pair-wise interactions between histone fold domain (HFD)-containing TAFs are likely to occur in the native yTFIID complex. Most of the HFD-containing TAFs have been found in two distinct lobes, thus revealing an unexpected and novel molecular organization of TFIID.

Amino Acid Motifs↗

PubMed2002

Expression of FLAG fusion proteins in insect cells: application to the multi-subunit transcription/DNA repair factor TFIIH.

The multi-subunit transcription/DNA repair factor TFIIH was used as a model system to show that the expression of FLAG fusion proteins in insect cells constitutes a versatile tool for both structural and functional investigations. In the present study, we have constructed recombinant baculoviruses expressing the four core TFIIH subunits fused at their N-terminus to the FLAG peptide. Using these recombinant viruses we have established protocols based on anti-FLAG immunoaffinity chromatography that allow the systematic analysis of pairwise interaction within multiprotein complexes and have developed a double tag strategy (FLAG and hexahistidine tags) for the identification and purification of stable TFIIH subcomplexes. A simple purification procedure was developed that leads to the isolation of recombinant TFIIH containing the full set of subunits. The purified recombinant TFIIH was shown to be active in a transcription assay and to be structurally homologous to the endogenous complex by electron microscopy and image analysis.

Animals↗