Site-specific labeling of Saccharomyces cerevisiae ribosomes for single-molecule manipulations.
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.
Petrov
Alexey
A
Department of Structural Biology and Stanford Magnetic Resonance Laboratory, Stanford University School of Medicine, Stanford, CA 94305-5126, USA.
Puglisi
Joseph D
JD
D005456:Fluorescent Dyes / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D015345:Oligonucleotide Probes / Q000737:chemistry*; D014176:Protein Biosynthesis; D012335:RNA, Ribosomal / Q000737:chemistry; D054683:Ribosome Subunits, Large, Eukaryotic / Q000737:chemistry*; D054682:Ribosome Subunits, Small, Eukaryotic / Q000737:chemistry*; D012270:Ribosomes / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D012335:RNA, Ribosomal