Escherichia coli RNA Polymerase Activity Observed Using Atomic Force Microscopy

Abstract

Fluid tapping-mode atomic force microscopy (AFM) was used to observe Escherichia coli RNA polymerase (RNAP) transcribing two different linear double-stranded (ds) DNA templates. The transcription process was detected by observing the translocation of the DNA template by RNAP on addition of ribonucleoside 5'-triphosphates (NTPs) in sequential AFM images. Stalled ternary complexes of RNAP, dsDNA and nascent RNA were adsorbed onto a mica surface and imaged under continuously flowing buffer. On introduction of all four NTPs, we observed some DNA molecules being pulled through the RNAP, some dissociating from the RNAP and others which did not move relative to the RNAP. The transcription rates were observed to be approximately 0.5-2 bases/s at our NTP concentrations, approximately 5 microM. The RNA transcripts were not unambiguously imaged in fluid. However, in experiments using a small single-stranded (ss) circular DNA template, known as a rolling circle, transcripts up to 1 or 2 microns long could be observed with tapping mode AFM once the samples were dried and imaged in air. This confirmed our observations of the transcriptional activity of RNAP adsorbed onto mica. This work illustrates that the development of tapping-mode in fluid has made it possible to use AFM to follow biological processes at the molecular level and get new insights about the variability of activity of individual molecules bound to a surface.

Single-Molecule Studies of RNA Polymerase: Motoring Along

“…Longer RNA transcripts could occasionally be visualized in TEC images, as well (76). However, the angles measured between RNA and DNA arms proved to be inconsistent with the currently modeled location of nucleic acids in the crystal structures, perhaps because of confounding surface interactions with the RNA, which is substantially less rigid than DNA, or because of the difficulties inherent in imaging a three-dimensional structure in two dimensions (77).…”

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].…”

Mechanisms of DNA binding determined in optical tweezers experiments

“…Once the spring constant of the tip is determined, picoNewton force measurements are possible, although the force resolution of the instrument is typically a bit poorer ($5-10 pN) than optical experiments. 7,13 This technique also has the advantage of rapid data collection as a complete stretch/relax cycle may be collected in seconds.…”