Osmolytes stimulate the reconstitution of functional 50S ribosomes from in vitro transcripts of Escherichia coli 23S rRNA.
Functional Escherichia coli 50S ribosomal subunits can be reconstituted from their natural rRNA and protein components. However, when the assembly is performed with in vitro-transcribed 23S rRNA, the reconstitution efficiency is diminished by four orders of magnitude. We tested a variety of chemical chaperones (compounds that are typically used for protein folding), putative RNA chaperones (proteins) and ribosome-targeted antibiotics (small-molecule ligands) that might be reasoned to aid in folding and assembly. Addition of the osmolyte trimethylamine-oxide (TMAO) and the ketolide antibiotic telithromycin (HMR3647) to the reconstitution stimulates its efficiency up to 100-fold yielding a substantially improved system for the in vitro analysis of mutant ribosomes.
Semrad
Katharina
K
Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Green
Rachel
R
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D002241:Carbohydrates / Q000494:pharmacology; D002364:Caseins / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics*; D048628:Ketolides; D008055:Lipids / Q000494:pharmacology; D018942:Macrolides; D008274:Magnesium / Q000378:metabolism; D008744:Methylamines / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009154:Mutation; D010458:Peptidyl Transferases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D014674:Plant Proteins, Dietary / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012338:RNA, Ribosomal, 23S; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D000900:Anti-Bacterial Agents; D002241:Carbohydrates; D002364:Caseins; D048628:Ketolides; D008055:Lipids; D018942:Macrolides; D008744:Methylamines; D014674:Plant Proteins, Dietary; D012333:RNA, Messenger; D012338:RNA, Ribosomal, 23S; C080652:Osmolite; D012343:RNA, Transfer; D010458:Peptidyl Transferases; C005855:trimethyloxamine; D008274:Magnesium; C106791:telithromycin