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Osmolytes stimulate the reconstitution of functional 50S ribosomes from in vitro transcripts of Escherichia coli 23S rRNA

Published online by Cambridge University Press:  24 April 2002

KATHARINA SEMRAD  and
RACHEL GREEN
KATHARINA SEMRAD
Affiliation:
Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
RACHEL GREEN
Affiliation:
Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
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Abstract

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.

Keywords

in vitro reconstitutionribosome assemblyRNA foldingtelithromycintrimethylamine oxide (TMAO)
Type
Research Article
Information
RNA , Volume 8 , Issue 4 , April 2002 , pp. 401 - 411
Copyright
2002 RNA Society

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