The antibiotic kasugamycin mimics mRNA nucleotides to destabilize tRNA binding and inhibit canonical translation initiation

Schluenzen, Frank; Takemoto, Chie; Wilson, Daniel N.; Kaminishi, T.; Harms, Joerg; Hanawa-Suetsugu, Kyoko; Szaflarski, Witold; Kawazoe, Masahito; Shirouzu, Mikako; Nierhaus, Knud H.; Yokoyama, Shigeyuki; Fucini, Paola

doi:10.1038/nsmb1145

Cited by 124 publications

(131 citation statements)

References 45 publications

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“…In helix 24a, U793 is in the space normally occupied by the methylated adenosines, and the two adjacent bases, A792 and A794, show small shifts. These bases comprise part of the kasugamycin binding site (Schluenzen et al 2006;Schuwirth et al 2006), and their movement is consistent with the finding that a base substitution, A794G, confers kasugamycin resistance (Vila-Sanjurjo et al 1999). Thus, as previously predicted (Schluenzen et al 2006;Schuwirth et al 2006), kasugamycin resistance due to lack of methylation most likely results from indirect effects on the conformation of helix 24a in the vicinity of bases directly involved in kasugamycin binding.…”

Section: Loss Of Helix Packing Interactionssupporting

confidence: 63%

Modification of 16S ribosomal RNA by the KsgA methyltransferase restructures the 30S subunit to optimize ribosome function

DeMi̇rci̇

Murphy²,

Belardinelli³

et al. 2010

RNA

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All organisms incorporate post-transcriptional modifications into ribosomal RNA, influencing ribosome assembly and function in ways that are poorly understood. The most highly conserved modification is the dimethylation of two adenosines near the 39 end of the small subunit rRNA. Lack of these methylations due to deficiency in the KsgA methyltransferase stimulates translational errors during both the initiation and elongation phases of protein synthesis and confers resistance to the antibiotic kasugamycin. Here, we present the X-ray crystal structure of the Thermus thermophilus 30S ribosomal subunit lacking these dimethylations. Our data indicate that the KsgA-directed methylations facilitate structural rearrangements in order to establish a functionally optimum subunit conformation during the final stages of ribosome assembly.

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Section: Loss Of Helix Packing Interactionssupporting

confidence: 63%

Modification of 16S ribosomal RNA by the KsgA methyltransferase restructures the 30S subunit to optimize ribosome function

DeMi̇rci̇

Murphy²,

Belardinelli³

et al. 2010

RNA

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show abstract

“…In the small ribosomal subunit, antibiotics act upon a variety of sites where the drugs may affect binding of tRNAs, displace mRNA, or interfere with translocation (11)(12)(13)(14)(15)(16). In a striking contrast, the distribution of sites of antibiotic action in the large ribosomal subunit is highly constrained.…”

mentioning

confidence: 92%

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

Yassin¹,

Mankin

2007

Journal of Biological Chemistry

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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.

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“…This antibiotic acts by destabilizing fMet-tRNA fMet binding to the 30S-mRNA preinitiation complex. 17 Moreover, kasugamycin induces the formation of 61S ribosomal particles in E. coli cells. The 61S ribosome lacks several 30S proteins, among which is S1, and is unable to translate leadered transcripts, but it is still proficient in leaderless mRNA translation.…”

Section: A Whole-cell Assay For Specific Inhibitors Of Translation Inmentioning

confidence: 99%

A Whole-Cell Assay for Specific Inhibitors of Translation Initiation in Bacteria

2015

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The bacterial translational apparatus is an ideal target for the search of new antibiotics. In fact, it performs an essential process carried out by a large number of potential subtargets for antibiotic action. Moreover, it is sufficiently different in several molecular details from the apparatus of Eukarya and Archaea to generally ensure specificity for the bacterial domain. This applies in particular to translation initiation, which is the most different step in the process. In bacteria, the 30S ribosomal subunit directly binds to the translation initiation region, a site within the messenger RNA (mRNA) 5′-untranslated region (5′-UTR). 30S binding is mediated by the interaction of both the 16S ribosomal RNA and the ribosomal protein S1 with specific regions of the mRNA 5′-UTR. An alternative, S1-independent pathway is enjoyed by leaderless mRNAs (i.e., transcripts devoid of a 5′-UTR). We have developed a simple fluorescence-based whole-cell assay in Escherichia coli to find inhibitors of the canonical S1-dependent translation initiation pathway. The assay has been set up both in a common E. coli laboratory strain and in a strain with an outer membrane permeability defect. Compared with other whole-cell assays for antibacterials, the major advantages of the screen described here are high sensitivity and specificity.

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The antibiotic kasugamycin mimics mRNA nucleotides to destabilize tRNA binding and inhibit canonical translation initiation

Cited by 124 publications

References 45 publications

Modification of 16S ribosomal RNA by the KsgA methyltransferase restructures the 30S subunit to optimize ribosome function

Modification of 16S ribosomal RNA by the KsgA methyltransferase restructures the 30S subunit to optimize ribosome function

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

A Whole-Cell Assay for Specific Inhibitors of Translation Initiation in Bacteria

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