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Biophys J. 2005 Sep;89(3):1909-19. Epub 2005 Jun 10.

Mechanical studies of single ribosome/mRNA complexes.

Author information

1
Pennsylvania Muscle Institute, Department of Bioengineering, and Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

Abstract

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.

PMID:
15951374
PMCID:
PMC1366694
DOI:
10.1529/biophysj.104.056283
[Indexed for MEDLINE]
Free PMC Article

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