DNA-Protein complexes spread on N2-discharged carbon film and characterized by molecular weight and its projected distribution. Nucleosome formation by an in Vitro transcription system

  • Hough P
  • Mastrangelo I
  • Wall J
 et al. 
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Abstract

Omission of stain and shadow and the use of uncoated thin carbon film provide unparalleled contrast and resolution for protein-nucleic acid complexes studied by scanning transmission electron microscopy. A new technique, electrical discharge of the film in an atmosphere of pure N2, provides the required adhesion and spreading. Molecular weight determination by electron scattering achieves nearly its minimum possible error under these circumstances, as shown by test measurement of Escherichia coli RNA polymerase bound to bacteriophage T7 DNA. DNA contour length is within a few per cent of form B length and shows a relative standard deviation of 2 to 3%, several-fold smaller than Kleinschmidt spreading for short fragments. These techniques have contributed to a demonstration of nucleosome formation on a fragment of adenovirus serotype 2 DNA during brief incubation with a soluble extract of HeLa cells known to give selective and accurate transcription by RNA polymerase II. An example indicates the effectiveness of this approach for study of polymerase subunits and protein factors involved in transcription initiation. © 1982.

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Authors

  • Paul V.C. Hough

  • Iris A. Mastrangelo

  • Joseph S. Wall

  • James F. Hainfeld

  • Martha N. Simon

  • James L. Manley

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