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Humanization of bacterial 16S rRNA decoding region

Applicant Sander Peter
Number 100780
Funding scheme Project funding (Div. I-III)
Research institution Institut für Medizinische Mikrobiologie Universität Zürich
Institution of higher education University of Zurich - ZH
Main discipline Medical Microbiology
Start/End 01.10.2003 - 31.12.2007
Approved amount 161'990.00
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Keywords (7)

ribosomal RNA; ribosome; protein synthesis; structure-function relationship; drug target; aminoglycoside resistance; selectivity

Lay Summary (English)

Lead
Lay summary
Structural and genetic studies on prokaryotic ribosomes have provided important insights into fundamental aspects of protein synthesis and the interaction of the ribosome with drugs affecting translation. Comparable mechanistic studies in eukaryotes are mainly hampered by the absence of both high-resolution crystal structures and efficient genetic models. In the project reported herein, we developed a genetic model for efficient rRNA gene replacement in bacteria. Deletion of all chromosomal rRNA genes and complementation with a single set of rRNA genes on a single-copy plasmid provided a genetic model not only for site-directed mutagenesis of specific rRNA nucleotides, but also for the exchange of entire functional rRNA domains.To study the interaction of aminoglycoside antibiotics with human cytosolic and mitochondrial ribosomes, we replaced the drug binding site in bacterial 16S rRNA with its human counterparts, resulting in bacterial hybrid ribosomes with a fully functional human rRNA decoding site. Various in-vivo and in-vitro assays demonstrated that hybrid ribosomes carrying the rRNA decoding site of human cytosolic ribosomes show pronounced resistance to aminoglycoside antibiotics, equivalent to that of rabbit reticulocyte ribosomes. Compared to the cytosolic hybrid, the mitochondrial hybrid showed distinct susceptibility to aminoglycoside antibiotics.The experimental system established within this project allowed us to investigate structure-function relationships in rRNA and to study the mechanisms determining selectivity and toxicity of aminoglycoside antibiotics. Our findings also suggest that phylogenetically variable components of the ribosome, other than the rRNA-binding site, do not affect aminoglycoside susceptibility of the protein-synthesis machinery. The activities of the hybrid ribosomes indicate that helix 44 of the rRNA decoding site behaves as an autonomous domain, which can be exchanged between ribosomes of different phylogenetic domains for study of function.
Direct link to Lay Summary Last update: 21.02.2013

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