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Negamycin interferes with decoding and translocation by simultaneous interaction with rRNA and tRNA.

ABSTRACT: Negamycin (NEG) is a ribosome-targeting antibiotic that exhibits clinically promising activity. Its binding site and mode of action have remained unknown. We solved the structure of the Thermus thermophilus ribosome bound to mRNA and three tRNAs, in complex with NEG. The drug binds to both small and large ribosomal subunits at nine independent sites. Resistance mutations in the 16S rRNA unequivocally identified the binding site in the vicinity of the conserved helix 34 (h34) in the small subunit as the primary site of antibiotic action in the bacterial and, possibly, eukaryotic ribosome. At this site, NEG contacts 16S rRNA as well as the anticodon loop of the A-site tRNA. Although the NEG site of action overlaps with that of tetracycline (TET), the two antibiotics exhibit different activities: while TET sterically hinders binding of aminoacyl-tRNA to the ribosome, NEG stabilizes its binding, thereby inhibiting translocation and stimulating miscoding.

SUBMITTER: Polikanov YS

PROVIDER: S-EPMC4334386 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Negamycin interferes with decoding and translocation by simultaneous interaction with rRNA and tRNA.

Polikanov Yury S YS Szal Teresa T Jiang Fuyan F Gupta Pulkit P Matsuda Ryoichi R Shiozuka Masataka M Steitz Thomas A TA Vázquez-Laslop Nora N Mankin Alexander S AS

Molecular cell 20141009 4

Negamycin (NEG) is a ribosome-targeting antibiotic that exhibits clinically promising activity. Its binding site and mode of action have remained unknown. We solved the structure of the Thermus thermophilus ribosome bound to mRNA and three tRNAs, in complex with NEG. The drug binds to both small and large ribosomal subunits at nine independent sites. Resistance mutations in the 16S rRNA unequivocally identified the binding site in the vicinity of the conserved helix 34 (h34) in the small subunit ...[more]

PMID: 25306922

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Project description:Escherichia coli contains seven rRNA operons, each consisting of the genes for three rRNAs (16S, 23S and 5S rRNA in this order) and one or two tRNA genes in the spacer between 16S and 23S rRNA genes and one or two tRNA genes in the 3' proximal region. All of these rRNA and tRNA genes are transcribed from two promoters, P1 and P2, into single large precursors that are afterward processed to individual rRNAs and tRNAs by a set of RNases. In the course of Genomic SELEX screening of promoters recognized by RNA polymerase (RNAP) holoenzyme containing RpoD sigma, a strong binding site was identified within 16S rRNA gene in each of all seven rRNA operons. The binding in vitro of RNAP RpoD holoenzyme to an internal promoter, referred to the promoter of riRNA (an internal RNA of the rRNA operon), within each 16S rRNA gene was confirmed by gel shift assay and AFM observation. Using this riRNA promoter within the rrnD operon as a representative, transcription in vitro was detected with use of the purified RpoD holoenzyme, confirming the presence of a constitutive promoter in this region. LacZ reporter assay indicated that this riRNA promoter is functional in vivo. The location of riRNA promoter in vivo as identified using a set of reporter plasmids agrees well with that identified in vitro. Based on transcription profile in vitro and Northern blot analysis in vivo, the majority of transcript initiated from this riRNA promoter was estimated to terminate near the beginning of 23S rRNA gene, indicating that riRNA leads to produce the spacer-coded tRNA. Under starved conditions, transcription of the rRNA operon is markedly repressed to reduce the intracellular level of ribosomes, but the levels of both riRNA and its processed tRNAGlu stayed unaffected, implying that riRNA plays a role in the continued steady-state synthesis of tRNAs from the spacers of rRNA operons. We then propose that the tRNA genes organized within the spacers of rRNA-tRNA composite operons are expressed independent of rRNA synthesis under specific conditions where further synthesis of ribosomes is not needed.

Project description:tRNAs play a critical role in mRNA decoding, and posttranscriptional modifications within tRNAs drive decoding efficiency and accuracy. The types and positions of tRNA modifications in model bacteria have been extensively studied, and tRNA modifications in a few eukaryotic organisms have also been characterized and localized to particular tRNA sequences. However, far less is known regarding tRNA modifications in archaea. While the identities of modifications have been determined for multiple archaeal organisms, Haloferax volcanii is the only organism for which modifications have been extensively localized to specific tRNA sequences. To improve our understanding of archaeal tRNA modification patterns and codon-decoding strategies, we have used liquid chromatography and tandem mass spectrometry to characterize and then map posttranscriptional modifications on 34 of the 35 unique tRNA sequences of Methanocaldococcus jannaschii A new posttranscriptionally modified nucleoside, 5-cyanomethyl-2-thiouridine (cnm5s2U), was discovered and localized to position 34. Moreover, data consistent with wyosine pathway modifications were obtained beyond the canonical tRNAPhe as is typical for eukaryotes. The high-quality mapping of tRNA anticodon loops enriches our understanding of archaeal tRNA modification profiles and decoding strategies.IMPORTANCE While many posttranscriptional modifications in M. jannaschii tRNAs are also found in bacteria and eukaryotes, several that are unique to archaea were identified. By RNA modification mapping, the modification profiles of M. jannaschii tRNA anticodon loops were characterized, allowing a comparative analysis with H. volcanii modification profiles as well as a general comparison with bacterial and eukaryotic decoding strategies. This general comparison reveals that M. jannaschii, like H. volcanii, follows codon-decoding strategies similar to those used by bacteria, although position 37 appears to be modified to a greater extent than seen in H. volcanii.

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Negamycin interferes with decoding and translocation by simultaneous interaction with rRNA and tRNA.

Publications

Negamycin interferes with decoding and translocation by simultaneous interaction with rRNA and tRNA.

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