Preparation and characterization of fluorescent 50S ribosomes. Specific labeling of ribosomal proteins L7/L12 and L10 of Escherichia coli.
So that the topographic and dynamic properties of the L7/L12--L10 complex in the 50S ribosome of Escherichia coli could be studied, methods and reagents were developed in order to introduce fluorescent groups at specific positions of these proteins. In the case of L7/L12, this was done by attaching an aldehyde group to Lys-51 of the protein by using 4-(4-formylphenoxy)butyrimidate or by converting the amino terminus of L12 into an aldehyde group by periodate oxidation. Subsequent reaction of the aldehyde groups with newly developed hydrazine derivatives of fluorescein and coumarin resulted in specifically labeled L7/L12 derivatives. L10 was modified at the single cysteine residue with N-[7-(dimethylamino)-4-methylcoumarinyl]maleimide. The fluorescent proteins L10 and L7/L12 could be reconstituted into 50S ribosomes. The resulting specifically labeled 50S ribosomes show 25--100% activity in elongation factor G dependent GTPase as well as in polyphenylalanine synthesis. The fluorescent properties of the labeled 50S ribosomes show that these fluorescent derivatives are suitable for energy transfer studies.
D004926:Escherichia coli / Q000032:analysis* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000138:chemical synthesis; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D000080882:Ribosomal Protein L10; D012269:Ribosomal Proteins / Q000302:isolation & purification*; D012270:Ribosomes / Q000032:analysis*; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D010446:Peptide Fragments; D012269:Ribosomal Proteins; C016700:ribosomal protein L7-L12; D020963:GTP Phosphohydrolase-Linked Elongation Factors