Imaging of T7 RNA polymerase elongation complexes by atomic force microscopy

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Abstract

Atomic force microscopy was used to visualize the complexes of bacteriophage T7 RNA polymerase (RNAP) with a DNA template during transcription. A 1414-bp fragment of linearized pGEMEX DNA was used as a template; the fragment contained the T7 promoter and terminator asymmetrically located at the template ends. Images of stable complexes of T7 RNAP and template terminal fragments were obtained for single molecules. Individual template DNA molecules bound with two or three T7 RNAP molecules, which corresponded to transcription initiation, elongation, and termination, and complexes containing RNA transcripts were imaged under conditions preventing nonspecific binding. The results suggest that T7 RNAP binds initially to the DNA template terminal fragment located near the promoter in order to exclude skipping the promoter site.

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