Oriented, Active Escherichia coli RNA Polymerase: An Atomic Force Microscope Study

Abstract

Combining a system for binding proteins to surfaces (Sigal, G. B., C. Bamdad, A. Barberis, J. Strominger, and G. M. Whitesides. 1996. Anal. Chem. 68:490-497) with a method for making ultraflat gold surfaces (Hegner, M., P. Wagner, and G. Semenza. 1993. Surface Sci. 291:39-46 1993) has enabled single, oriented, active Escherichia coli RNA polymerase (RNAP) molecules to be imaged under aqueous buffer using tapping-mode atomic force microscopy (AFM). Recombinant RNAP molecules containing histidine tags (hisRNAP) on the C-terminus were specifically immobilized on ultraflat gold via a mixed monolayer of two different omega-functionalized alkanethiols. One alkanethiol was terminated in an ethylene-glycol (EG) group, which resists protein adsorption, and the other was terminated in an N-nitrilotriacetic acid (NTA) group, which binds the histidine tag through two coordination sites with a nickel ion. AFM images showed that these two alkanethiols phase-segregate. Specific binding of the hisRNAP molecules was followed in situ by injecting proteins directly into the AFM fluid cell. The activity of the hisRNAP bound to the NTA groups was confirmed with a 42-base circular single-stranded DNA template (rolling circle), which the RNAP uses to produce huge RNA transcripts. These transcripts were imaged in air after the samples were rinsed and dried, since RNA also has low affinity for the EG-thiol and cannot be imaged under the buffers we used.

Mechanical Unfolding of the Beet Western Yellow Virus −1 Frameshift Signal

“…Atomic force microscopy (AFM), single molecule fluorescence microscopy, and optical tweezers have made it possible to measure the displacements, velocities, and forces that molecular motors produce (60)(61)(62). In particular, optical tweezers have proven useful for studying molecular motors, because the forces they produce are of the same order of magnitude (pN) as those exerted by molecular motors.…”

Preparation of flat gold terraces for protein chip developments

“…Stripping can be also obtained through a chemical treatment using THF. This last possibility presents also limitations especially in terms of surface contamination by mica flasks 6 .…”

Recent developments in the site‐specific immobilization of proteins onto solid supports

“…3 The use of recombinant affinity tags addresses the orientation issue. 2,[16][17][18][19][20][21][22][23][24][25] However, in most cases, the interactions of the tags are reversible and not stable over time. 24,[26][27][28][29] Site-specific covalent immobilization, on the other hand, allows the proteins to be arranged in a definite, controlled fashion.…”