Direct Observation of One-Dimensional Diffusion and Transcription by Escherichia coli RNA Polymerase

Abstract

The dynamics of nonspecific and specific Escherichia coli RNA polymerase (RNAP)-DNA complexes have been directly observed using scanning force microscopy operating in buffer. To this end, imaging conditions had to be found in which DNA molecules were adsorbed onto mica strongly enough to be imaged, but loosely enough to be able to diffuse on the surface. In sequential images of nonspecific complexes, RNAP was seen to slide along DNA, performing a one-dimensional random walk. Heparin, a substance known to disrupt nonspecific RNAP-DNA interactions, prevented sliding. These observations suggest that diffusion of RNAP along DNA constitutes a mechanism for accelerated promoter location. Sequential images of single, transcribing RNAP molecules were also investigated. Upon addition of 5 microM nucleoside triphosphates to stalled elongation complexes in the liquid chamber, RNAP molecules were seen to processively thread their template at rates of 1.5 nucleotide/s in a direction consistent with the promoter orientation. Transcription assays, performed with radiolabeled, mica-bound transcription complexes, confirmed this rate, which was about three times smaller than the rate of complexes in solution. This assay also showed that the pattern of pause sites and the termination site were affected by the surface. By using the Einstein-Sutherland friction-diffusion relation the loading force experienced by RNAP due to DNA-surface friction is estimated and discussed.

Single-Molecule Studies of RNA Polymerase: Motoring Along

“…Other estimates of diffusion rates have been obtained using time-resolved AFM to acquire sequential images every 100 seconds for RNAP diffusing along λ DNA. The position of RNAP on the DNA varied from one image to the next, consistent with a random walk in one dimension with a diffusion rate of 10 1 nm 2 /s (45,46), which is again too low to explain bulk search data.…”

High-speed atomic force microscopy coming of age

“…A group including one of the inventors of AFM (i.e., Gerd Binnig) also attempted to image the dynamic processes of live cells being infected by virus [27] and binding of antibody to the S-layer [28]. After the introduction of the tapping mode, DNA-involved dynamic processes were studied by Paul Hansma's group and Carlos Bustamante's group; DNA digestion by DNAase [29] , binding of RNA polymerase to DNA [30], transcription process [31], and diffusion of RNA polymerase along DNA strands [32]. Other types of dynamic processes studied were digestion of collagen I by collagenase [33] and closing of nuclear pores upon exposure to 5% CO 2 [34].…”

Protein–DNA Electrostatics

“…It is interesting to note that, despite its good performances, this experience is open to multiple interpretations [9], and its results are difficult to reproduce by simple models [38]. Alternative techniques as atomic force microscopy [39] and fluorescence microscopy [40] (Figure 4) allow a direct visualization of the protein movement.…”

“…In the case of protein-DNA systems, protein and DNA can either be fixed adsorbed onto the surface, loosely enough to be able to diffuse on it [39]. Despite the very high spatial resolution, this technique was initially limited by a low temporal resolution: tens of seconds between two images.…”