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Mechanical Studies of Single Ribosome/mRNA Complexes

Francesco Vanzi ^*, Yasuharu Takagi , Henry Shuman ^*, Barry S. Cooperman  and Yale E. Goldman ^*

^* Pennsylvania Muscle Institute, Department of Bioengineering, and Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania

Correspondence: Address reprint requests to Yale E. Goldman, Pennsylvania Muscle Institute, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6083. Tel.: 215-898-4017; Fax: 215-898-2653; E-mail: goldmany{at}mail.med.upenn.edu.

Methodology was developed for specifically anchoring Escherichia coli 70S ribosomes onto a chemically modified, cysteine-reactive glass surface. Immobilized ribosomes maintain the capability of binding a polyuridylic acid (poly(U)) template, enabling investigation of mechanical properties of individual ribosome-poly(U) complexes using laser tweezers. Streptavidin-coated polystyrene microspheres bound specifically to the biotinylated 3' end of long (up to 10,000 bases) poly(U) strands. A novel optical method was built to control the position of the laser trap along the microscope optical axis at 2 nm resolution, facilitating measurement of the force-extension relationship for poly(U). Some immobilized ribosome-poly(U) complexes supported 100 pN of force applied at the 3' end of the mRNA. Binding of N-acetylated Phe-tRNA^Phe, an analog of the initiator fMet-tRNA^Met, enhanced the population of complexes that could withstand high forces. The persistence length of poly(U) RNA homopolymer, modeled as a worm-like chain, was found to be 0.79 ± 0.05 nm and the backbone elasticity was 900 ± 140 pN, similar to values for single-stranded DNA.

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