Erythromycin- and Chloramphenicol-Induced Ribosomal Assembly Defects Are Secondary Effects of Protein Synthesis Inhibition

Siibak, Triinu; Peil, Lauri; Xiong, Liqun; Mankin, Alexander S.; Rèmme, Jaanus; Tenson, Tanel

doi:10.1128/aac.00870-08

Cited by 74 publications

(67 citation statements)

References 64 publications

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“…S13, treatment with the antibiotics led to increase in accumulation of 16S rRNA precursor. Biogenesis defects upon treatment with translation inhibitors were also seen earlier (34,35). It was proposed that an imbalance between rRNA transcription and sufficiency of r-proteins due to translational inhibition led to the defect.…”

Section: Significancementioning

confidence: 75%

An evolutionarily conserved element in initiator tRNAs prompts ultimate steps in ribosome maturation

Shetty

Varshney

2016

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

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Ribosome biogenesis, a complex multistep process, results in correct folding of rRNAs, incorporation of >50 ribosomal proteins, and their maturation. Deficiencies in ribosome biogenesis may result in varied faults in translation of mRNAs causing cellular toxicities and ribosomopathies in higher organisms. How cells ensure quality control in ribosome biogenesis for the fidelity of its complex function remains unclear. Using Escherichia coli, we show that initiator tRNA (i-tRNA), specifically the evolutionarily conserved three consecutive GC base pairs in its anticodon stem, play a crucial role in ribosome maturation. Deficiencies in cellular contents of i-tRNA confer cold sensitivity and result in accumulation of ribosomes with immature 3′ and 5′ ends of the 16S rRNA. Overexpression of i-tRNA in various strains rescues biogenesis defects. Participation of i-tRNA in the first round of initiation complex formation licenses the final steps of ribosome maturation by signaling RNases to trim the terminal extensions of immature 16S rRNA.cold sensitivity | ribosome biogenesis | 3GC base pairs | RNase PH/RNAse R | 16S rRNA maturation R ibosomes are large and the most complex of the molecular machines in cells, using up to 40% of the cellular energy for their biogenesis (1). Ribosome biogenesis is a multistep process involving a coordinated network of RNA-RNA, RNA-protein, and protein-protein interactions. The process begins with the transcription of rRNA, its processing, nucleoside modifications, structural rearrangements, synthesis, and interaction of ribosomal proteins (r-proteins) (2). In cells, RNA processing enzymes, modification systems, and chaperone-like factors facilitate ribosome biogenesis. Any deficiencies in this process may produce ribosomes that fail to maintain the fidelity of protein synthesis and cause cellular toxicity/ death (3). Deficiencies in biogenesis factors impart cold sensitivity to Escherichia coli and affect bacterial drug resistance and virulence. In humans, genetic defects resulting in imperfect ribosome biogenesis are the cause of various ribosomopathies (4-7). Thus, a better understanding of ribosome biogenesis is a fundamental requirement to develop antimicrobials and various therapies (8-12).Of the two ribosomal subunits in E. coli, the small subunit contains a 1,542 nucleotide long rRNA (16S rRNA) along with 21 r-proteins, whereas the large subunit contains two rRNAs of 2,904 (23S rRNA) and 120 (5S rRNA) nucleotide lengths along with 33 r-proteins. The three rRNAs are synthesized as parts of a single transcript and the assembly of r-proteins begins cotranscriptionally. As the assembly advances, the precursor rRNA is cleaved by RNase III into immature units of 16S rRNA (as 17S), 23S rRNA (as 25S), and 5S rRNA (as 9S) rRNAs. The 17S rRNA retains unprocessed extensions of 110 and 33 nucleotides on the 5′ and 3′ ends, respectively. Trimming of the extensions by RNase G and other RNases matures 16S rRNA. Incorporation of r-proteins occurs in three stages. The primary r-proteins interact di...

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Section: Significancementioning

confidence: 75%

An evolutionarily conserved element in initiator tRNAs prompts ultimate steps in ribosome maturation

Shetty

Varshney

2016

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

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“…Nevertheless, this accumulation could be explained as a consequence of impaired protein synthesis due to a shortage of mature ribosomes in these cells. Indeed, it has been reported that disturbances in protein synthesis arising from exposure to a 50S subunittargeting antibiotic, such as puromycin, erythromycin, or chloramphenicol, also induce accumulation of 17S rRNA (Hosokawa and Nomura 1965;Himeno et al 2004;Siibak et al 2009). Thus it is conceivable that the structure of the immature 30S subunit purified from the DYjeQ strain may not represent the binding substrate for YjeQ but rather for RbfA.…”

Section: Discussionmentioning

confidence: 99%

Understanding ribosome assembly: the structure of in vivo assembled immature 30S subunits revealed by cryo-electron microscopy

Jomaa¹,

Stewart²,

Martín‐Benito³

et al. 2011

RNA

111

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Four decades after early in vitro assembly studies demonstrated that ribosome assembly is a controlled process, our understanding of ribosome assembly is still incomplete. Just as structure determination has been so important to understanding ribosome function, so too will it be critical to sorting out the assembly process. Here, we used a viable deletion in the yjeQ gene, a recognized ribosome assembly factor, to isolate and structurally characterize immature 30S subunits assembled in vivo. These small ribosome subunits contained unprocessed 17S rRNA and lacked some late ribosomal proteins. Cryo-electron microscopy reconstructions revealed that the presence of precursor sequences in the rRNA induces a severe distortion in the 39 minor domain of the subunit involved in the decoding of mRNA and interaction with the large ribosome subunit. These findings suggest that rRNA processing events induce key local conformational changes directing the structure toward the mature assembly. We concluded that rRNA processing, folding, and the entry of tertiary r-proteins are interdependent events in the late stages of 30S subunit assembly. In addition, we demonstrate how studies of emerging assembly factors in ribosome biogenesis can help to elucidate the path of subunit assembly in vivo.

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“…To determine the effects of translation on nucleoid exclusion, we used the antibiotic erythromycin, which inhibits translation and induces an accumulation of ribosomal precursor particles (17,18). First, we determined the effects of translation on the size of the nucleoid by tracking HU proteins in untreated and erythromycin-treated cells (Fig.…”

Section: Free Ribosomal Subunit Precursors Are Partially Excluded Fromentioning

confidence: 99%

Single-particle tracking reveals that free ribosomal subunits are not excluded from the Escherichia coli nucleoid

Sanamrad

Persson

Fange

et al. 2014

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

183

228

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Biochemical and genetic data show that ribosomes closely follow RNA polymerases that are transcribing protein-coding genes in bacteria. At the same time, electron and fluorescence microscopy have revealed that ribosomes are excluded from the Escherichia coli nucleoid, which seems to be inconsistent with fast translation initiation on nascent mRNA transcripts. The apparent paradox can be reconciled if translation of nascent mRNAs can start throughout the nucleoid before they relocate to the periphery. However, this mechanism requires that free ribosomal subunits are not excluded from the nucleoid. Here, we use single-particle tracking in living E. coli cells to determine the fractions of free ribosomal subunits, classify individual subunits as free or mRNA-bound, and quantify the degree of exclusion of bound and free subunits separately. We show that free subunits are not excluded from the nucleoid. This finding strongly suggests that translation of nascent mRNAs can start throughout the nucleoid, which reconciles the spatial separation of DNA and ribosomes with cotranscriptional translation. We also show that, after translation inhibition, free subunit precursors are partially excluded from the compacted nucleoid. This finding indicates that it is active translation that normally allows ribosomal subunits to assemble on nascent mRNAs throughout the nucleoid and that the effects of translation inhibitors are enhanced by the limited access of ribosomal subunits to nascent mRNAs in the compacted nucleoid.nucleoid exclusion | transcription-translation coupling | antibiotics | single-molecule tracking | single-molecule imaging I n bacteria, translation often starts soon after the ribosomebinding site emerges from the RNA exit channel of the RNA polymerase. The transcribing RNA polymerase is then closely followed by translating ribosomes in such a way that the overall transcription elongation rate is tightly controlled by the translation rate (1). This coupling between transcription and translation of nascent mRNAs is important for regulatory mechanisms that respond to the formation of gaps between the transcribing RNA polymerases and the trailing ribosomes. Such gaps may, for example, allow the formation of secondary structures that allow RNA polymerases to proceed through transcription termination sites (2). The gaps may also allow the transcription termination factor Rho to access the nascent mRNAs and terminate transcription (3).Bacterial 70S ribosomes are formed when large 50S subunits and small 30S subunits assemble on mRNAs. Electron and fluorescence microscopy have revealed that ribosomes are excluded from the Escherichia coli nucleoid (4-6), but this spatial separation of DNA and ribosomes has not yet been reconciled with cotranscriptional translation. The paradox can be resolved if translation of nascent mRNAs can start throughout the nucleoid before they relocate to the periphery (7). However, this mechanism requires that free ribosomal subunits are not excluded from the nucleoid.To determine whether free r...

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Erythromycin- and Chloramphenicol-Induced Ribosomal Assembly Defects Are Secondary Effects of Protein Synthesis Inhibition

Cited by 74 publications

References 64 publications

An evolutionarily conserved element in initiator tRNAs prompts ultimate steps in ribosome maturation

An evolutionarily conserved element in initiator tRNAs prompts ultimate steps in ribosome maturation

Understanding ribosome assembly: the structure of in vivo assembled immature 30S subunits revealed by cryo-electron microscopy

Single-particle tracking reveals that free ribosomal subunits are not excluded from the Escherichia coli nucleoid

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