Site-specific labeling of Saccharomyces cerevisiae ribosomes for single-molecule manipulations

Alexey Petrov 1 Joseph D. Puglisi 0 1 0 Stanford Magnetic Resonance Laboratory, Stanford University School of Medicine , Stanford, CA 94305-5126, USA 1 Department of Structural Biology Site-specific labeling of Escherichia coli ribosomes has allowed application of single-molecule fluorescence spectroscopy and force methods to probe the mechanism of translation. To apply these approaches to eukaryotic translation, eukaryotic ribosomes must be specifically labeled with fluorescent labels and molecular handles. Here, we describe preparation and labeling of the small and large yeast ribosomal subunits. Phylogenetically variable hairpin loops in ribosomal RNA are mutated to allow hybridization of oligonucleotides to mutant ribosomes. We demonstrate specific labeling of the ribosomal subunits, and their use in single-molecule fluorescence and force experiments. - Single-molecule methods have extensively investigated the mechanisms of translation in prokaryotes. By removing space and time averaging inherent to bulk systems, single-molecule approaches allow studies of heterogeneous and asynchronous systems. Protein synthesis is a multistep repetitive process that rapidly becomes asynchronous as ribosomes progress through multiple steps of elongation thus challenging application of conventional biochemical and biophysical techniques. To overcome this obstacle, single-molecule fluorescence has been applied to probe interactions between ribosomes, tRNA and translation factors, and to monitor conformational dynamics of the ribosome. Introduction of the fluorescent dye pairs that can undergo Fo rster resonance energy transfer (FRET) into the ribosome and its ligands revealed conformational changes occurring in ribosome and tRNA as ribosome progresses through initiation, elongation and termination (14). FRET pairs incorporated into ribosomal subunits reported on the conformational and structural dynamics of the ribosomal particle and revealed new steps and kinetic intermediates (58). Fluorescent labeling of the ribosomal subunits permitted continuous observation of ribosome dynamics through initiation followed by multiple rounds of elongation using single-molecule detection, avoiding the problem of rapid desynchronization of the ribosome population; these observations were only possible at a single-molecule level (7,9). Specific labeling of the ribosomal particles also allowed application of the force methods. Changes in mechanical stability of the mRNA:ribosome complexes in response to bound tRNA ligands revealed mechanism of ShineDalgarno clearance transition to elongation (10). Subsequent work by Bustamante group allowed observation of individual ribosome progression along mRNA (11). Thus, manipulation of bacterial ribosomes was central to allow detailed singlemolecule investigation of mechanisms of prokaryotic translation. Two general strategies for labeling ribosomal particles have been applied to prokaryotic ribosomes. The well-studied self-assembly of ribosomal particles from isolated protein and RNA allows introduction of the fluorescent labels into ribosomal proteins. Purified proteins are labeled using maleimide dye chemistry at genetically introduced single cysteine residues. Ribosomal particles are then assembled using the labeled proteins (12). Labeled versions of several ribosomal proteins, such as L1 or L11 can be incorporated to preassembled ribosomes that are missing the unlabeled versions of the proteins, created biochemically or genetically. Alternatively, metastable hairpins that readily anneal with labeled oligonucleotides can be genetically introduced into rRNA (13). Phylogenetic analysis guides the sites of mutation that do not disrupt ribosomal function. This allows placement of the fluorescent labels and molecular handles necessary for force measurements into ribosomal subunits. Genetic systems allow selection of pure populations of functionally mutant ribosomes that are subsequently labeled by hybridization with dye-linked oligonucleotides. Both labeling approaches have been used to explore dynamics of the bacterial ribosomal particle [Reviewed in (14)]. Eukaryotic translational mechanism is highly complex and regulated. Translation initiation involves 50-cap recognition, scanning and start codon selection. Alternative mechanisms allow distinct translational responses. Application of bulk methods has revealed the main steps of initiation, but precise molecular mechanisms remain obscure. The application of single-molecule methods will allow investigation of eukaryotic translation at singlemolecule level at nanometer scale. Complex maturation and assembly process of eukaryotic ribosomes complicates labeling of the individual eukaryotic ribosomal proteins with subsequent incorporation into ribosomal particle. Here, we exploited an rRNA modification approach and describe the construction and characterization of yeast ribosomes bearing labeling hairpins in surface exposed regions of rRNA. We demonstrate specific labeling of yeast ribosomes with fluorescently labeled oligonucleotides, and application of these ribosomes to singlemolecule fluorescence and force experiments. MATERIALS AND METHODS Strains, media, reagents and molecular methods Escherichia coli strain DH5a was used for cloning and to amplify plasmids. Yeast media contained 2% galactose instead of glucose; drug concentrations were as follows: doxycycline, 10 mg/ml; and hygromycin B, 300 mg/ml. Transformations of yeast strains were performed according to an alkaline cation protocol and yeast cells were grown at 30 C. Yeast strain pJD1314 lacking RDN operons (MATa ade2-1 ura3-1 trp1-1 his3-11 leu2-3, can1-100 D rDNA::his3::hisG+[pNOY353 (GAL7-RDN37 RDN5 TRP1 2 AMP)] [L-A HN M1]) and pJD694 plasmid containing RDN35 under tetracycline repressible promoter were kindly provided by J. D. Dinman (15). Total yeast RNA was isolated by acid phenol extraction and treated with RNase-free DNase (QIAGEN, Valencia, CA, USA). The Titan One-Tube RT-PCR system (Roche) was used for reverse transcriptionPCR (RTPCR). Ribosome mutagenesis Mutations were introduced into 18S and 25S rRNA of Saccharomyces cerevisiae. Two-step megaprimer PCR was used to incorporate labeling hairpins into pJD694 (URA3) plasmid carrying 35S rRNA under a tetracycline repressible promoter. Corresponding primers are listed in Supplementary Table S1. The resulting plasmids were transformed into pJD1314 S. cerevisiae strain, where RDN operons were deleted and supplied from 2 mm TRP1-carrying plasmid under galactose inducible promoter (15). Transformants were grown on Ura, Trp, Gal, Dox media for 10 days and then streaked twice on the Ura, Trp, Gal media for 3 days to establish stable expression of the mutant rRNA. Subsequently, strains were replica plated on Ura media, to turn of transcription of wild-type rRNA. Strains were replica-plated four more times on the Ura media to induce spontaneous loss of the TRP1-containing plasmid. The resultin (...truncated)