Mechanical studies of single ribosome/mRNA complexes.
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.
Vanzi
Francesco
F
Pennsylvania Muscle Institute, Department of Bioengineering, and Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Takagi
Yasuharu
Y
Shuman
Henry
H
Cooperman
Barry S
BS
Goldman
Yale E
YE
D001669:Biochemical Phenomena; D001671:Biochemistry; D001696:Biomechanical Phenomena; D001703:Biophysics / Q000379:methods*; D019921:Biotinylation; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D007834:Lasers; D008863:Microspheres; D015233:Models, Statistical; D010441:Peptide Chain Elongation, Translational; D011072:Poly U / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D011137:Polystyrenes / Q000737:chemistry*; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry*; D019809:Streptavidin / Q000494:pharmacology*; D013499:Surface Properties; D013696:Temperature; D013997:Time Factors
D004277:DNA, Single-Stranded; D011108:Polymers; D011137:Polystyrenes; D012333:RNA, Messenger; D011072:Poly U; D012313:RNA; D004247:DNA; D019809:Streptavidin; D003545:Cysteine