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

Using total internal reflection fluorescence microscopy, we have directly o bserved individual interactions of single RNA polymerase molecules with a s ingle molecule of lambda-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 similar to 0.66 s(-1), which was more tha n severalfold smaller than the rate at other positions. The association rat e constant for the slow dissociation sites was 9.2 x 10(2) 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-ri ch regions. RNA polymerase molecules on the fast dissociation sites underwe nt linear diffusion (sliding) along DNA. The binding to the slow dissociati on sites was greatly enhanced when DNA was released to a relaxed state, sug gesting that the binding depended on the strain exerted on the DNA. The pre sent method is potentially applicable to the examination of a wide variety of protein-nucleic acid interactions, especially those involved in the proc ess of transcription.