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

Katharina Semrad; Rachel Green

doi:10.1017/S1355838202029722

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

Katharina Semrad, Rachel Green

School of Medicine

Research output: Contribution to journal › Article › peer-review

37 Scopus citations

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.

Original language	English (US)
Pages (from-to)	401-411
Number of pages	11
Journal	RNA
Volume	8
Issue number	4
DOIs	https://doi.org/10.1017/S1355838202029722
State	Published - 2002

Keywords

In vitro reconstitution
RNA folding
Ribosome assembly
Telithromycin
Trimethylamine oxide (TMAO)

ASJC Scopus subject areas

Molecular Biology

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10.1017/S1355838202029722

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

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.",

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T1 - Osmolytes stimulate the reconstitution of functional 50S ribosomes from in vitro transcripts of Escherichia coli 23S rRNA

AU - Semrad, Katharina

AU - Green, Rachel

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N2 - 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.

AB - 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.

KW - In vitro reconstitution

KW - RNA folding

KW - Ribosome assembly

KW - Telithromycin

KW - Trimethylamine oxide (TMAO)

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

Abstract

Keywords

ASJC Scopus subject areas

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