Oriented, active Escherichia coli RNA polymerase: An atomic force microscope study

Combining a system for binding proteins to surfaces (Sigal, G. B., C. Bamda d, A. Barberis, J. Strominger, and G. NI. Whitesides. 1996, Anal. Chem. 68: 490-497) with a method for making ultraflat gold surfaces (Hegner M., P. Wa gner, and G. Semenza. 1993. Surface Sci. 291:39-46 1993) has enabled single , oriented, active Escherichia coil RNA polymerase (RNAP) molecules to be i maged under aqueous buffer using tapping-mode atomic force microscopy (AFM) . Recombinant RNAP molecules containing histidine tags (hisRNAP) on the C-t erminus were specifically immobilized on ultraflat gold via a mixed monolay er of two different omega-functionalized alkanethiols. One alkanethiol was terminated in an ethylene-glycol (EG) group, which resists protein adsorpti on, 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. Specif ic binding of the hisRNAP molecules was followed in situ by injecting prote ins 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 te mplate (rolling circle), which the RNAP uses to produce huge RNA transcript s. These transcripts were imaged in air after the samples were rinsed and d ried, since RNA also has low affinity for the EG-thiol and cannot be imaged under the buffers we used.