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Biomedical subjects

C Bustamante

Publications and source records attributed to C Bustamante.

At least 73 records · Page 4Linked to original sources
PubMed1995

Determination of heat-shock transcription factor 2 stoichiometry at looped DNA complexes using scanning force microscopy.

Gene activation frequently requires an array of proteins bound to sites distal to the transcription start site. The assembly of these protein-bound sites into specialized nucleoprotein complexes is a prerequisite for transcriptional activation. Structural analysis of these higher order complexes will provide crucial information for understanding the mechanisms of gene activation. We have used both electron microscopy and scanning force microscopy to elucidate the structure of complexes formed between DNA and heat-shock transcription factor (HSF) 2, a human heat-shock transcriptional activator that binds DNA as a trimer. Electron microscopy reveals that HSF2 will bring together distant DNA sites to create a loop. We show that this association requires only the DNA binding and trimerization domains of HSF2. Metal shadowing techniques used for electron microscopy obscure details of these nucleoprotein structures. Greatly increased resolution was achieved by directly imaging the complexes in the scanning force microscope, which reveals that at least two trimers are required for the association of HSF2-bound DNA sites.

DNA↗
PubMed1994

Following the assembly of RNA polymerase-DNA complexes in aqueous solutions with the scanning force microscope.

The capability of the scanning force microscope (SFM) to image molecules in aqueous buffers has opened the exciting possibility of following processes of molecular assembly in real time and in near-physiological environments. This capability is demonstrated in this paper by following the assembly process of RNA polymerase-DNA complexes. DNA fragments deposited on mica and imaged in Hepes/MgCl2 are shown before and after Escherichia coli RNA polymerase holoenzyme is injected in the SFM liquid chamber. The protein can recognize and bind to these DNA fragments within several seconds after injection, suggesting that the protein and the DNA retain their native configuration after deposition and during SFM imaging. A time-lapse sequence depicting the process of assembly of RNA polymerase-DNA complexes is shown. These results represent the first step for acquiring the capabilities to monitor complex biomolecular processes as they take place in ionic solutions and to characterize their spatial organization.

Bacterial Proteins↗
PubMed1994

DNA bending by Cro protein in specific and nonspecific complexes: implications for protein site recognition and specificity.

Scanning force microscopy was used to resolve lambda Cro protein when bound as a single dimer or multiple dimers to its three operator (OR) sites. The bend angles induced by binding of Cro to specific and nonspecific sites were determined and are 69 degrees +/- 11 degrees for specific and 62 degrees +/- 23 degrees for nonspecific complexes. Bending of the nonspecific sites is advantageous for a protein such as Cro that bends its specific site, because it increases the binding specificity of the protein and it can be used by the protein to sample contacts required for the recognition of its target sequence. It is proposed here that bending of nonspecific DNA may be a general property among DNA binding proteins that bend their specific sites.

Base Sequence↗
PubMed1994

Three-dimensional structure of extended chromatin fibers as revealed by tapping-mode scanning force microscopy.

Unfixed chicken erythrocyte chromatin fibers in very low salt have been imaged with a scanning force microscope operating in the tapping mode in air at ambient humidity. These images reveal a three-dimensional organization of the fibers. The planar "zig-zag" conformation is rare, and extended "beads-on-a-string" fibers are seen only in chromatin depleted of histones H1 and H5. Glutaraldehyde fixation reveals very similar structures. Fibers fixed in 10 mM salt appear somewhat more compacted. These results, when compared with modeling studies, suggest that chromatin fibers may exist as irregular three-dimensional arrays of nucleosomes even at low ionic strength.

Animals↗
PubMed1994

Linker DNA accessibility in chromatin fibers of different conformations: a reevaluation.

New studies on chromatin fiber morphology, using the technique of scanning force microscopy (SFM), have caused us to reexamine recent analysis of nuclease digestion of chromatin. Chicken erythrocyte chromatin fibers, glutaraldehyde-fixed at 0, 10, and 80 mM NaCl, were imaged with the help of SFM. The chromatin fibers possessed a loose three-dimensional 30-nm structure even in the absence of added salt. This structure slightly condensed upon addition of 10 mM NaCl, and highly compacted, irregularly segmented fibers were observed at 80 mM NaCl. This sheds new light upon our previously reported analysis of the kinetics of digestion by soluble and membrane-immobilized micrococcal nuclease [Leuba, S. H., Zlatanova, J. & van Holde, K. (1994) J. Mol. Biol. 235, 871-880]. While the low-ionic-strength fibers were readily digested, the highly compacted structure formed at 80 mM NaCl was refractory to nuclease attack, implying that the linkers were fully accessible in the low-ionic-strength conformation but not in the condensed fibers. We now find that cleavage of the linker DNA by a small molecule, methidiumpropyl-EDTA-Fe(II), proceeds for all types of conformations at similar rates. Thus, steric hindrance is responsible for the lack of accessibility to micrococcal nuclease in the condensed fiber. Taken in total the data suggest that reexamination of existing models of chromatin conformation is warranted.

Animals↗
PubMed1994

Interaction of water-soluble porphyrins with single- and double-stranded polyribonucleotides.

CD and uv-visible absorption studies with several tetracationic water-soluble porphyrin derivatives show that some of these species can serve as probes to discriminate between A- and B-conformational forms of single-stranded polynucleotides. It is also observed that these porphyrins can participate in the formation of double helices by forming transient intermediate complexes en-route to duplex formation.

Chemical Phenomena↗
PubMed1993

Masking generates contiguous segments of metal-coated and bare DNA for scanning tunneling microscope imaging.

To date, no microscopic methods are available to confirm scanning tunneling microscope (STM) images of DNA. The difficulties encountered in repeating these images may be attributed to inadequate distribution of molecules on the substrate, poor adhesion to the substrate, or the low conductivity of the molecules. However, these factors are difficult to assess in an STM experiment where they may act simultaneously. A method to isolate these factors involves partly masking the deposited molecules before coating them with a conductive film to produce adjacent segments of coated and bare DNA after the mask is removed. The coated DNA segments are conductive and mechanically stable to allow easy identification of DNA by the STM. Furthermore, the path of a molecule can be traced from a coated to an uncoated region to test STM imaging of bare DNA. Masked preparations of DNA deposited on platinum/carbon-coated mica and highly oriented pyrolytic graphite were examined with a tunneling current 1000 times lower than the usual nanoamps. The tip apparently displaces molecules adsorbed to graphite to preclude imaging whereas more stably bound DNA on platinum/carbon-coated mica appears in reversed contrast.

Aluminum Silicates↗
PubMed1993

Evidence of DNA bending in transcription complexes imaged by scanning force microscopy.

Complexes of Escherichia coli RNA polymerase with DNA containing the lambda PL promoter have been deposited on mica and imaged in air with a scanning force microscope. The topographic images reveal the gross spatial relations of the polymerase relative to the DNA template. The DNA appears bent in open promoter complexes containing RNA polymerase bound to the promoter and appears more severely bent in elongation complexes in which RNA polymerase has synthesized a 15-nucleotide transcript. This difference could be related to the conformational changes that accompany the maturation of open promoter complexes into elongation complexes and suggests that formation of the elongation complex involves a considerable modification of the spatial relations between the polymerase and the DNA template.

DNA↗
PubMed1993

Towards a molecular description of pulsed-field gel electrophoresis.

An improved understanding of the molecular processes of migration and reorientation of DNA molecules undergoing conventional and pulsed-field gel electrophoresis (PFGE) should provide the basis for the rational optimization of these techniques. This review of common pulsed-field methods emphasizes the range of their applications as well as their current limitations. The molecular mechanisms involved in electrophoresis, as they are beginning to emerge from a combination of direct observations using a microscope and computer-modeling studies, are also discussed.

Computer Simulation↗
PubMed1992

Direct mechanical measurements of the elasticity of single DNA molecules by using magnetic beads.

Single DNA molecules were chemically attached by one end to a glass surface and by their other end to a magnetic bead. Equilibrium positions of the beads were observed in an optical microscope while the beads were acted on by known magnetic and hydrodynamic forces. Extension versus force curves were obtained for individual DNA molecules at three different salt concentrations with forces between 10(-14) and 10(-11) newtons. Deviations from the force curves predicted by the freely jointed chain model suggest that DNA has significant local curvature in solution. Ethidium bromide and 4',6-diamidino-2-phenylindole had little effect on the elastic response of the molecules, but their extent of intercalation was directly measured. Conversely, the effect of bend-inducing cis-diamminedichloroplatinum (II) was large and supports the hypothesis of natural curvature in DNA.

Chemical Phenomena↗
PubMed1992

Circular DNA molecules imaged in air by scanning force microscopy.

Routine and reproducible imaging of DNA molecules in air with the scanning force microscope (SFM) has been accomplished. Circular molecules of plasmid DNA were deposited onto red mica and imaged under various relative humidities. In related experiments, the first images of the Escherichia coli RNA polymerase-DNA complex have also been obtained. This has been possible by (1) the use of specially modified SFM tips with a consistent radius of curvature of 10 nm or less, to minimize the amount of image distortion introduced by the finite dimensions of commercially available tips, (2) the optimization of a method to deposit and bind DNA molecules to the mica surface in a stable fashion, and (3) careful control of the sample humidity, to prevent solvation of the molecules and detachment from the surface by the scanning tip or stylus. Contact forces in the range of a few nanonewtons are routinely possible in air and in the presence of residual humidity. The spatial resolution of the images appears determined by the radius of curvature of the modified styli, which can be estimated directly from the apparent widths of the DNA molecules in the images.

Air↗
PubMed1992

Theoretical framework for the interpretation of STM images of adsorbates.

A theoretical formalism for the interpretation of STM images of adsorbates is developed by approaching the calculation of the observed current as a transport problem in quantum statistical mechanics. The STM configuration is treated as a system of three groups of states--the substrate, the adsorbate and the tip--in contact with a thermal reservoir, with which it exchanges energy. A new definition of current is introduced, and shown to be related to that given in the traditional transfer Hamiltonian approach. The transport instrument used for the description is the stochastic Liouville equation, known to have the advantage of allowing the incorporation of thermal effects as well as arbitrary degree of coherence in the quantum transport. Some preliminary calculations of STM images of simple adsorbate models are presented.

Computer Simulation↗
PubMed1992

Electrodeposition procedure of E. coli RNA polymerase onto gold and deposition of E. coli RNA polymerase onto mica for observation with scanning force microscopy.

Molecules of the transcriptional enzyme E. coli RNA polymerase (RNAP) have been deposited using three different deposition methods: (1) passive adsorption onto gold, (2) electrochemical adsorption onto gold and (3) adsorption onto mica. In all cases SFM imaging was straightforward and reliable, and surface coverage by the protein varied with deposition conditions as expected. To determine the nature of the electrochemical treatment on the gold substrate, cyclic voltammetry was performed with various chemical solutions. Finally, a comparison is made between the SFM images of RNAP obtained with these methods and STM images obtained earlier. Both STM and SFM show strikingly similar results; however, heights and widths of individual molecules differ.

Adsorption↗