Download The Length of the 5`-Leader of Escherichia coli tRNA

Molecular insights into ribosome functions by atomic mutagenesis
Norbert Polacek
Innsbruck Biocenter, Medical University Innsbruck, Division of Genomics and
RNomics, Austria
Biochemical and crystallographic studies revealed the ribosome as an RNAenzyme. 23S rRNA is the main component of the large ribosomal subunit and is involved
in catalyzing the two principal chemical reactions of protein synthesis, peptide bond
formation and peptidyl tRNA hydrolysis during the elongation and termination phases of
translation, respectively. Despite accumulating biochemical and structural insights, the
catalytic mechanisms of 23S rRNA are still not fully understood in molecular terms.
The aim of our research is to gain molecular insight into ribosomal functions by
employing a newly developed in vitro reconstitution assay of ribosomal particles. By this
approach single functional groups or even single atom modifications can be incorporated
at specific positions of the 23S rRNA. This ‘atomic mutagenesis’ approach significantly
enlarges the pool of chemically distinct residues that can be placed specifically in the
ribosome compared to standard mutagenesis. Equipped with this new molecular tool we
have identified distinct rRNA backbone groups to be crucial for catalyzing peptide bond
formation as well as for promoting peptidyl-tRNA hydrolysis.
Following peptide bond formation, the two ribosome-bound tRNAs have to be
translocated from the A- and P-sites to the P- and E-sites, respectively. This movement is
mediated by the GTPase EF-G. Using the atomic mutagenesis approach, we have recently
revealed the exocyclic N6 amino group of an universally conserves adenosine residue in
the so called sarcin-ricin loop of 23S rRNA as potential trigger of EF-G GTPase.