Single-molecule imaging of RNA polymerase-DNA interactions in real time

Yoshie Harada, Takashi Funatsu, Katsuhiko Murakami, Yoshikazu Nonoyama, Akira Ishihama, Toshio Yanagida

Research output: Contribution to journalArticle

217 Scopus citations

Abstract

Using total internal reflection fluorescence microscopy, we have directly observed individual interactions of single RNA polymerase molecules with a single molecule of λ-phage DNA suspended in solution by optical traps. The interactions of RNA polymerase molecules were not homogeneous along DNA. They dissociated slowly from the positions of the promoters and sequences common to promoters at a rate of ~0.66 s-1, which was more than severalfold smaller than the rate at other positions. The association rate constant for the slow dissociation sites was 9.2 X 102 bp-1 M-1 s-1. The frequency of binding to the fast dissociation sites was dependent on the A-T composition; it was larger in the AT-rich regions than in the GC-rich regions. RNA polymerase molecules on the fast dissociation sites underwent linear diffusion (sliding) along DNA. The binding to the slow dissociation sites was greatly enhanced when DNA was released to a relaxed state, suggesting that the binding depended on the strain exerted on the DNA. The present method is potentially applicable to the examination of a wide variety of protein-nucleic acid interactions, especially those involved in the process of transcription.

Original languageEnglish (US)
Pages (from-to)709-715
Number of pages7
JournalBiophysical journal
Volume76
Issue number2
DOIs
StatePublished - 1999

All Science Journal Classification (ASJC) codes

  • Biophysics

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