REPORTProtein synthesis by single ribosomesFRANCESCO VANZI,1,4SERGUEI VLADIMIROV,2CHARLOTTE R. KNUDSEN,3YALE E. GOLDMAN,1andBARRY S. COOPERMAN21Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6083, USA2Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323, USA3Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus C, DenmarkABSTRACTThe ribosome is universally responsible for synthesizing proteins by translating the genetic code transcribed in mRNA into anamino acid sequence. Ribosomes use cellular accessory proteins, soluble transfer RNAs, and metabolic energy to accomplish theinitiation, elongation, and termination of peptide synthesis. In translocating processively along the mRNA template during theelongation cycle, ribosomes act as supramolecular motors. Here we demonstrate that ribosomes adsorbed on a surface, as formechanical or spectroscopic studies, are capable of polypeptide synthesis and that tethered particle analysis of fluorescentbeads connected to ribosomes via polyuridylic acid can be used to estimate the rate of polyphenylalanine synthesis by individualribosomes. This work opens the way for application of biophysical techniques, originally developed for the classical motorproteins, to the understanding of protein biosynthesis.Keywords: Ribosome; single molecule; tethered particle; poly(U); poly(Phe)INTRODUCTIONAstonishing progress on the structure of the ribosome andits accessory factors has been obtained from cryo-electronmicroscopy (Stark et al. 2000; Frank 2001) and X-ray dif-fraction studies (Ban et al. 2000; Yusupov et al. 2001; Ra-makrishnan 2002; Yonath 2002). The kinetics of the elon-gation cycle have been elucidated by steady-state and tran-sient kinetics studies (Rodnina et al. 2000). Although theenergy required for formation of the peptide bond comesfrom the ester bond of tRNA charged with its cognateamino acid, the remarkable rate of translation (10–20 pep-tide bonds/sec; Kjeldgaard and Gausing 1974; Kennell andRiezman 1977), fidelity of amino acid selection, and main-tenance of the reading frame during translocation (∼10−4error rate; Loftfield and Vanderjagt 1972; Kurland 1992)require the GTPase activities of two G-protein elongationfactors, EF-Tu and EF-G. The mechanisms of action ofthese factors are not fully understood because the structuralchanges they undergo and the mechanical events they fa-cilitate have not been elucidated on functioning ribosomes.Single-molecule techniques have been used to measuredirectly the elementary events of production of force anddisplacement by molecular motors (Kinosita 1999; Mehta etal. 1999; Ishijima and Yanagida 2001) and nucleic acid pro-cessing enzymes (Rich 1998; Wang et al. 1998; Strick et al.2000; Wuite et al. 2000), helping to elucidate their proper-ties and mechanisms. Because the sliding of the ribosomealong the mRNA template is a mechanical output, analo-gous single-molecule measurements should provide infor-mation relevant toward the understanding of the molecularmechanism of protein synthesis. For instance, applying anexternal force to the mRNA and measuring the relationshipbetween the rate of translocation and the force appliedcould help to distinguish between proposed mechanisms oftranslocation (Czworkowski and Moore 1997; Keller andBustamante 2000; Wintermeyer and Rodnina 2000).It has been shown previously that ribosomes adsorbed ona mica surface are competent to bind aminoacyl-tRNA inthe peptidyl transfer site (P-site) and to form a peptidebond with added puromycin (Sytnik et al. 1999). Here weextend this work by measuring bulk poly(Phe) synthesis onpoly(U)-programmed mica-bound ribosomes and detectingtranslocation of individual ribosome-bound poly(U) tem-plates using optical microscopy to examine mRNA-tetheredbeads.Reprint requests to: Barry S. Cooperman, Department of Chemistry,University of Pennsylvania, Philadelphia, PA 19104-6323, USA; e-mail:[email protected]; or Yale E. Goldman, Pennsylvania MuscleInstitute, University of Pennsylvania, Philadelphia, PA 19104-6083, USA;e-mail: [email protected]; fax: (215) 898-2653.Present address:4LENS–INFM, University of Florence, Florence, Italy.Abbreviations: poly(Phe), polyphenylalanine; poly(U), polyuridylic acid;TCA, trichloroacetic acid; TPM, tethered particle method.Article and publication are at http://www.rnajournal.org/cgi/doi/10.1261/rna.5800303.RNA (2003), 9:1174–1179. Published by Cold Spring Harbor Laboratory Press. Copyright © 2003 RNA Society.1174RESULTS AND DISCUSSIONWe investigated whether poly(U)-pro-grammed ribosomes, adsorbed nonspe-cifically to mica surfaces, could synthe-size TCA-precipitable poly([3H]-Phe)and translocate along poly(U) mRNA.Ribosomes were adsorbed to mica sam-ple chambers from solutions containing500 nM or 10 nM concentrations of 70Sparticles. These concentrations approxi-mate the conditions used in measuringpoly(Phe) synthesis in bulk solution andribosome activity in the microscopic ex-periments or poly(Phe) synthesis onmica, respectively. Quantification of ad-sorbed material was achieved using 70S particles labeledwith [32P]-phosphate. At both loading concentrations, alayer of stably bound ribosomes remained on the surfaceafter extensive washing, although removal of non-stablybound ribosomes required more washes at the lower ap-plied concentration. We obtained 1.5 ± 0.2 (n = 4) and0.020 ± 0.002 (n = 21; data not shown) pmole of boundribosomes/cm2for the higher and lower concentrations,respectively. These measurements indicate that at 500 nM,a multilayer of ribosomes (4–5 deep) is obtained, whereas at10 nM, a sparse distribution of ribosomes is achieved, with∼8% of the surface occupied. For the experiments describedbelow, the number of ribosomes present in solution due todetachment from the surface was negligible. Surface imagesof ribosomes adsorbed to mica obtained by tapping-modeatomic force microscopy (Fig. 1) also showed that the den-sity of tightly bound ribosomes is related to the initial con-centration applied to the surface. At 37 nM loading con-centration, the surface was covered with densely packedribosomes, often in clear multilayers. At ⱕ2.7 nM loading,a more sparse coverage was achieved, with single ribosomesgenerally isolated from one another.Mica-bound ribosomes synthesize poly(Phe) in an elon-gation-factor-dependent manner (Fig. 2A). On average,peptide
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