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[79] Electron microscopy of proteins bound to DNA

1980; Academic Press; Linguagem: Inglês

10.1016/s0076-6879(80)65081-2

1557-7988

Robert Schleif, Jay Hirsh,

Enzyme Structure and Function

Publisher Summary This chapter discusses the techniques for the electron microscopy of proteins bound to DNA. Microscopy is used to determine the positions of the bound proteins or protein complexes on DNA molecules and the conformation of DNA to which repressor or RNA polymerase had bound. Direct observation of proteins bound to DNA helps in understanding the mechanisms of gene regulation and nucleic acid metabolism. The chapter uses the approach of binding from a solution onto positively charged hydrophilic carbon grids and staining with uranyl formate, to determine the conditions necessary for RNA polymerase binding to promoters, repressor binding to operators, and formation of induction complexes on the arabinose operon. Advantages of these techniques are their parsimonious consumption of biological macromolecules and high information yield of a direct visual method. The absence of extraneous proteins or of contrast enhancement by shadowing permits high resolution and thus, occasionally, morphological information of proteins may be extracted.