An Unexpected Type of Ribosomes Induced by Kasugamycin: A Look into Ancestral Times of Protein Synthesis?

Kaberdina, Anna Chao; Szaflarski, Witold; Nierhaus, Knud H.; Moll, Isabella

doi:10.1016/j.molcel.2008.12.014

Cited by 108 publications

(123 citation statements)

References 53 publications

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“…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. 18 We have designed a simple whole-cell fluorescent screen to identify specific inhibitors of the S1-dependent pathway of bacterial translation initiation. As this process is both essential and specific for bacteria, such inhibitors would constitute promising hits in the research of new antibacterial drugs.…”

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

confidence: 99%

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

confidence: 99%

“…17,18 Several E. coli strains have been reported to be basically resistant to kasugamycin (minimal inhibitory concentration [MIC], ≥500 µg/mL), 28 probably because of poor cell entry, but the AS19 strain is sensitive to such an antibiotic (MIC between 75 and 100 µg/mL; data not shown).…”

Section: Evaluation Of the Assay Specificitymentioning

confidence: 99%

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

2015

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“…Leaderless mRNAs are devoid of 5= untranslated regions (mRNA leaders) and bear the AUG initiation codon at their 5= terminus or very close to it (reviewed in references 44 and 20). One of the features specific for leaderless mRNAs in E. coli is that they can be translated by ribosomes unable to form the initiation complexes with canonical leadered mRNAs, in particular, by the ribosomes devoid of the r-proteins S1 and S2 (6,18,21) and even some other r-proteins, as was shown for the cells treated with kasugamycin (45). Another peculiarity of the leaderless mRNAs is their capability of forming initiation complexes with 70S ribosomes (21,(46)(47)(48)(49), albeit the 30S-mediated initiation pathway cannot be completely ruled out either.…”

Section: Resultsmentioning

confidence: 94%

“…The mechanisms for leaderless mRNA translation in E. coli have so far been mainly studied by using either in vitro techniques or in vivo experiments with plasmids carrying the leaderless translational fusions with the lacZ reporter (18,21,(45)(46)(47)(48)(49)(50)(51)(52). Because the copy number of plasmids may vary depending on different factors, for in vivo experiments it would be preferable to use a singlecopy (chromosomal) genetic construction producing a leaderless transcript.…”

Section: Resultsmentioning

confidence: 99%

A Single Missense Mutation in a Coiled-Coil Domain of Escherichia coli Ribosomal Protein S2 Confers a Thermosensitive Phenotype That Can Be Suppressed by Ribosomal Protein S1

et al. 2013

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Ribosomal protein S2 is an essential component of translation machinery, and its viable mutated variants conferring distinct phenotypes serve as a valuable tool in studying the role of S2 in translation regulation. One of a few available rpsB mutants, rpsB1, shows thermosensitivity and ensures enhanced expression of leaderless mRNAs. In this study, we identified the nature of the rpsB1 mutation. Sequencing of the rpsB1 allele revealed a G-to-A transition in the part of the rpsB gene which encodes a coiled-coil domain of S2. The resulting E132K substitution resides in a highly conserved site, TKKE, a so-called N-terminal capping box, at the beginning of the second alpha helix. The protruding coiled-coil domain of S2 is known to provide binding with 16S rRNA in the head of the 30S subunit and, in addition, to interact with a key mRNA binding protein, S1. Molecular dynamics simulations revealed a detrimental impact of the E132K mutation on the coiled-coil structure and thereby on the interactions between S2 and 16S rRNA, providing a clue for the thermosensitivity of the rpsB1 mutant. Using a strain producing a leaderless lacZ transcript from the chromosomal lac promoter, we demonstrated that not only the rpsB1 mutation generating S2/S1-deficient ribosomes but also the rpsA::IS10 mutation leading to partial deficiency in S1 alone increased translation efficiency of the leaderless mRNA by about 10-fold. Moderate overexpression of S1 relieved all these effects and, moreover, suppressed the thermosensitive phenotype of rpsB1, indicating the role of S1 as an extragenic suppressor of the E132K mutation. Ribosomal protein (r-protein) S2 is essential for the translational machinery and is highly conserved across all kingdoms of life (S2 in prokaryotic-type ribosomes, S0 in yeast, and SA in higher eukaryotes), and yet its functions in protein synthesis are still poorly understood. It was suggested that prokaryotic S2 might be involved in stabilizing the Shine-Dalgarno (SD) helix docked in a chamber between the head and the platform (1), as well as in protecting the SD duplex at the early postinitiation step (2). However, this does not explain the vital function of S2 in organisms that do not exploit the SD interactions in translation initiation. S2 is one of the latest components identified in the 30S assembly (3). In Escherichia coli and most likely in other Gram-negative organisms, its association with the 30S particle is critical for binding the S1 r-protein that accomplishes the assembly of the 30S subunit that is fully competent in recruiting mRNA (4-6).Even though the structure of S2 has not yet been resolved for a free protein, it may be extracted from the high-resolution X-ray structure of the ribosome (7, 8). As the ribosome to be crystallized must be depleted of the r-protein S1, S2 (29.3 kDa) appears to be the largest r-protein in the crystal. It is located on the back of the 30S subunit at the hinge between the head and body (Fig. 1). Possessing an elongated bidomain structure, S2 forms several direct contacts w...

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“…The role of IFs in LL mRNA translation is not clearly defined, but IF2 enhances LL mRNA translation (Grill et al 2001) and IF3 discriminates against ribosome binding and translation of LL mRNA (Tedin et al 1999;O'Donnell and Janssen 2002;Udagawa et al 2004). Recent evidence reveals that ''61S'' ribosomes, resulting from kasugamycin treatment of cells, lack several 30S subunit proteins and are capable of selectively translating leaderless mRNAs (Kaberdina et al 2009). It is evident that the translation initiation pathway of LL mRNA differs from that of canonical mRNA.…”

Section: Introductionmentioning

confidence: 99%

A 5′-terminal phosphate is required for stable ternary complex formation and translation of leaderless mRNA in Escherichia coli

Giliberti¹,

O’Donnell²,

Etten³

et al. 2012

RNA

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The bacteriophage l's cI mRNA was utilized to examine the importance of the 59-terminal phosphate on expression of leadered and leaderless mRNA in Escherichia coli. A hammerhead ribozyme was used to produce leadered and leaderless mRNAs, in vivo and in vitro, that contain a 59-hydroxyl. Although these mRNAs may not occur naturally in the bacterial cell, they allow for the study of the importance of the 59-phosphorylation state in ribosome binding and translation of leadered and leaderless mRNAs. Analyses with mRNAs containing either a 59-phosphate or a 59-hydroxyl indicate that leaderless cI mRNA requires a 59-phosphate for stable ribosome binding in vitro as well as expression in vivo. Ribosome-binding assays show that 30S subunits and 70S ribosomes do not bind as strongly to 59-hydroxyl as they do to 59-phosphate containing leaderless mRNA and the tRNAdependent ternary complex is less stable. Additionally, filter-binding assays revealed that the 70S ternary complex formed with a leaderless mRNA containing a 59-hydroxyl has a dissociation rate (k off ) that is 4.5-fold higher compared with the complex formed with a 59-phosphate leaderless mRNA. Fusion to a lacZ reporter gene revealed that leaderless cI mRNA expression with a 59-hydroxyl was >100-fold lower than the equivalent mRNA with a 59-phosphate. These data indicate that a 59-phosphate is an important feature of leaderless mRNA for stable ribosome binding and expression.

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An Unexpected Type of Ribosomes Induced by Kasugamycin: A Look into Ancestral Times of Protein Synthesis?

Cited by 108 publications

References 53 publications

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

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

A Single Missense Mutation in a Coiled-Coil Domain of Escherichia coli Ribosomal Protein S2 Confers a Thermosensitive Phenotype That Can Be Suppressed by Ribosomal Protein S1

A 5′-terminal phosphate is required for stable ternary complex formation and translation of leaderless mRNA in Escherichia coli

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