Structural analysis of kasugamycin inhibition of translation

Schuwirth, B.S.; Day, J. Michael; Hau, C.W.; Janssen, Gary R.; Dahĺberg, Albert E.; Cate, J.H.D.; Vila-Sanjurjo, Antón

doi:10.1038/nsmb1150

Cited by 132 publications

(127 citation statements)

References 43 publications

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“…Unmethylated 30S subunits produced tetragonal crystals in the same space group, P4 1 2 1 2, that are obtained with fully methylated subunits from wild-type T. thermophilus, but crystallized slowly as small needles over a period of 1 yr, rather than in the 2 mo normally required for 30S subunits. Crystallization of unmethylated subunits was completely inhibited by kasugamycin, suggesting that T. thermophilus ksgA 30S subunits retain the ability to bind kasugamycin, as previously observed with an E. coli ksgA mutant (Schuwirth et al 2006).…”

Section: Resultssupporting

confidence: 58%

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: Resultssupporting

confidence: 58%

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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“…Similarly, we found that the steady-state level of lysC mRNA is increased significantly in the presence of the antibiotic kasugamycin (SI Appendix, Fig. S8B), which specifically blocks initiating ribosomes at the RBS (35). However, even in the complete absence of ribosome binding (RBS mutant), a similar lysine-dependent reduction of the mRNA level is still observed (approximately twofold as compared with the wildtype) (Fig.…”

Section: Discussionmentioning

confidence: 73%

Dual-acting riboswitch control of translation initiation and mRNA decay

Caron

Bastet

Lussier

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

158

150

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Riboswitches are mRNA regulatory elements that control gene expression by altering their structure in response to specific metabolite binding. In bacteria, riboswitches consist of an aptamer that performs ligand recognition and an expression platform that regulates either transcription termination or translation initiation. Here, we describe a dual-acting riboswitch from Escherichia coli that, in addition to modulating translation initiation, also is directly involved in the control of initial mRNA decay. Upon lysine binding, the lysC riboswitch adopts a conformation that not only inhibits translation initiation but also exposes RNase E cleavage sites located in the riboswitch expression platform. However, in the absence of lysine, the riboswitch folds into an alternative conformation that simultaneously allows translation initiation and sequesters RNase E cleavage sites. Both regulatory activities can be individually inhibited, indicating that translation initiation and mRNA decay can be modulated independently using the same conformational switch. Because RNase E cleavage sites are located in the riboswitch sequence, this riboswitch provides a unique means for the riboswitch to modulate RNase E cleavage activity directly as a function of lysine. This dual inhibition is in contrast to other riboswitches, such as the thiamin pyrophosphate-sensing thiM riboswitch, which triggers mRNA decay only as a consequence of translation inhibition. The riboswitch control of RNase E cleavage activity is an example of a mechanism by which metabolite sensing is used to regulate gene expression of single genes or even large polycistronic mRNAs as a function of environmental changes.gene regulation | RNA degradosome | translational control S ince the first demonstration that translation attenuation regulates the expression of the tryptophan operon (1), accumulating evidence has revealed the importance of posttranscriptional regulation in prokaryotes and eukaryotes alike. Posttranscriptional regulators include RNA molecules that operate through several mechanisms to control a wide range of physiological responses (2). Among newly identified RNA regulators are riboswitches, which are located in untranslated regions of several mRNAs and that modulate gene expression at the level of transcription, translation, or splicing (3). Riboswitches are highly structured regulatory domains that directly sense cellular metabolites such as amino acids, carbohydrates, coenzymes, and nucleobases. These genetic switches are composed of two modular domains consisting of an aptamer and an expression platform. The aptamer is involved in the specific recognition of the metabolite, and the expression platform is used to control gene expression by altering the structure of the mRNA. Recent bioinformatic analyses have reported the existence of several new RNA motifs exhibiting unconventional expression platforms (4, 5), suggesting that riboswitches using different regulation mechanisms are still likely to be discovered (6).The lysine riboswitch was first...

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“…In particular, the dimethylamine of m 6 2 A1519 is involved in medium and long-range repulsive interactions with the backbone of m 3 U1498 and the 2' O of C1520, while its conformation is mostly determined by the dimethylamine moiety of the adjacent m 6 2 A1518 which, in turn, is fixed by short repulsive interactions with O6 of G1517 and O4 of U793. The additional methyl groups of m 6 2 A1519 interact with A792, which provides part of the binding site for the antibiotic kasugamycin 15 , accounting for the resistance against kasugamycin upon demethylation of m 6 2 A1519 66 . Furthermore, m 6 2 A1518 and m 6 2 A1519 impact initiation 67 possibly via m 3 U1498 whose backbone interacts with m 6 2 A1519, while its modified base contacts the mRNA backbone.…”

Section: Extended Datamentioning

confidence: 99%

“…The P-site codon directly contacts m 4 Cm1402 and m 3 U1498 in 16S rRNA, which in turn are held in place by the bulky dimethylamine groups on m 6 2 A1519 and m 6 2 A1518. The network of long-range interactions provides the basis for the action of the antibiotic kasugamycin, which binds in the P site and requires dimethylation of m 6 2 A1519 for its function 15 . In the A site of the decoding centre, the aminoglycoside class of antibiotics directly binds to a monomethylated residue, m 5 C1407 in 16S rRNA, which is needed for optimum drug activity 16 (Extended Data Fig.…”

mentioning

confidence: 99%

Structure of the E. coli ribosome–EF-Tu complex at <3 Å resolution by Cs-corrected cryo-EM

Fischer

Neumann

Konevega

et al. 2015

Nature

364

443

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Single particle electron cryomicroscopy (cryo-EM) has recently made significant progress in high-resolution structure determination of macromolecular complexes due to improvements in electron microscopic instrumentation and computational image analysis. However, cryo-EM structures can be highly non-uniform in local resolution 1,2 and all structures available to date have been limited to resolutions above 3 Å 3,4 . Here we present the cryo-EM structure of the 70S ribosome from Escherichia coli in complex with elongation factor Tu, aminoacyl-tRNA and the antibiotic kirromycin at 2.65-2.9 Å resolution using spherical aberration (C s )-corrected cryo-EM. Overall, the cryo-EM reconstruction at 2.9 Å resolution is comparable to the best-resolved X-ray structure of the E. coli 70S ribosome 5 (2.8 Å ), but provides more detailed information (2.65 Å ) at the functionally important ribosomal core. The cryo-EM map elucidates for the first time the structure of all 35 rRNA modifications in the bacterial ribosome, explaining their roles in fine-tuning ribosome structure and function and modulating the action of antibiotics. We also obtained atomic models for flexible parts of the ribosome such as ribosomal proteins L9 and L31. The refined cryo-EM-based model presents the currently most complete high-resolution structure of the E. coli ribosome, which demonstrates the power of cryo-EM in structure determination of large and dynamic macromolecular complexes.Determining the structure of large, dynamic biological macromolecules at a uniformly high resolution provides a challenge both for X-ray crystallography and cryo-EM. Here we have used aberration-corrected cryo-EM in combination with extensive computational sorting to solve *These authors contributed equally to this work.

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Structural analysis of kasugamycin inhibition of translation

Cited by 132 publications

References 43 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

Dual-acting riboswitch control of translation initiation and mRNA decay

Structure of the E. coli ribosome–EF-Tu complex at <3 Å resolution by Cs-corrected cryo-EM

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