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

• Published in: Biophysical journal Vol. 76; no. 2; pp. 1024 - 1033
• Main Authors: Thomson, Neil H., Smith, Bettye L., Almqvist, Nils, Schmitt, Lutz, Kashlev, Mikhail, Kool, Eric T., Hansma, Paul K.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.1999; Biophysical Society
• Subjects: Bacteria; Biochemistry; DNA, Single-Stranded - metabolism; DNA-Directed RNA Polymerases - metabolism; DNA-Directed RNA Polymerases - ultrastructure; Enzymes; Escherichia coli - enzymology; Fysik; Gold; Microscopy, Atomic Force - methods; Nitrilotriacetic Acid - metabolism; Physics; Protein Binding; Recombinant Proteins - metabolism; Recombinant Proteins - ultrastructure; Ribonucleic acid; RNA; RNA - analysis; Sulfhydryl Compounds - metabolism; Transcription, Genetic - genetics
• ISSN: 0006-3495; 1542-0086; 1542-0086
• DOI: 10.1016/S0006-3495(99)77267-X

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 ω-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.