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J. Biol. Chem., Vol. 282, Issue 33, 24329-24342, August 17, 2007

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Potential New Antibiotic Sites in the Ribosome Revealed by Deleterious Mutations in RNA of the Large Ribosomal Subunit^*

From the Center for Pharmaceutical Biotechnology, University of Illinois, Chicago, Illinois 60607

The ribosome is the main target for antibiotics that inhibit protein biosynthesis. Despite the chemical diversity of the known antibiotics that affect functions of the large ribosomal subunit, these drugs act on only a few sites corresponding to some of the known functional centers. We have used a genetic approach for identifying structurally and functionally critical sites in the ribosome that can be used as new antibiotic targets. By using randomly mutagenized rRNA genes, we mapped rRNA sites where nucleotide alterations impair the ribosome function or assembly and lead to a deleterious phenotype. A total of 77 single-point deleterious mutations were mapped in 23 S rRNA and ranked according to the severity of their deleterious phenotypes. Many of the mutations mapped to familiar functional sites that are targeted by known antibiotics. However, a number of mutations were located in previously unexplored regions. The distribution of the mutations in the spatial structure of the ribosome showed a strong bias, with the strongly deleterious mutations being mainly localized at the interface of the large subunit and the mild ones on the solvent side. Five sites where deleterious mutations tend to cluster within discrete rRNA elements were identified as potential new antibiotic targets. One of the sites, the conserved segment of helix 38, was studied in more detail. Although the ability of the mutant 50 S subunits to associate with 30 S subunits was impaired, the lethal effect of mutations in this rRNA element was unrelated to its function as an intersubunit bridge. Instead, mutations in this region had a profound deleterious effect on the ribosome assembly.

Received for publication, April 12, 2007 , and in revised form, June 19, 2007.

^* This work was supported by National Institutes of Health Grant U19 AI56575. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Dept. of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Kasr El-Aini St., Cairo 11562, Egypt.

² To whom correspondence should be addressed: Center for Pharmaceutical Biotechnology, m/c 870, University of Illinois, 900 South Ashland Ave., Chicago, IL 60607. Fax: 312-413-9303; E-mail: shura{at}uic.edu.