Generation of Chemically Engineered Ribosomes for Atomic Mutagenesis Studies on Protein Biosynthesis

Overview

Journal Nat Protoc

Specialties Biology
Pathology
Science

Date 2011 Apr 30

PMID 21527916

Citations 25

Authors

Matthias D Erlacher

Anna Chirkova

Paul Voegele

Norbert Polacek

Affiliations

Soon will be listed here.

Abstract

The protocol describes the site-specific chemical modification of 23S rRNA of Thermus aquaticus ribosomes. The centerpiece of this 'atomic mutagenesis' approach is the site-specific incorporation of non-natural nucleoside analogs into 23S rRNA in the context of the entire 70S ribosome. This technique exhaustively makes use of the available crystallographic structures of the ribosome for designing detailed biochemical experiments aiming at unraveling molecular insights of ribosomal functions. The generation of chemically engineered ribosomes carrying a particular non-natural 23S rRNA residue at the site of interest, a procedure that typically takes less than 2 d, allows the study of translation at the molecular level and goes far beyond the limits of standard mutagenesis approaches. This methodology, in combination with the presented tests for ribosomal functions adapted to chemically engineered ribosomes, allows unprecedented molecular insight into the mechanisms of protein biosynthesis.

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