Creating ribosomes with an all-RNA 30S subunit P site

Hoang, Lee; Fredrick, Kurt; Noller, Harry F.

doi:10.1073/pnas.0405227101

Cited by 74 publications

(75 citation statements)

References 23 publications

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“…Overexpression of i-tRNA rescues the biogenesis defect caused either by the lack of 16S rRNA methylations in the P site (positions 966 and 967) or the C-terminal tail residues of S9 that affect i-tRNA binding (28,42). The i-tRNA could serve as a "template" for proper structuring of the P-site elements (particularly the rRNA) by establishing contacts via its 3GC pairs, and serve as a signal for final processing.…”

Section: Discussionmentioning

confidence: 99%

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: Discussionmentioning

confidence: 99%

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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“…The P-site features of 16S rRNA are omitted for clarity. The short and long portions of the tails that were subjected to chromosomal deletions [29] are shown in yellow and red, respectively.…”

Section: Involvement Of Ribosomal Proteinsmentioning

confidence: 99%

“…To test the functional importance of the protein components of the 30S P site, the tails of S9 and S13 were deleted, both singly and in combination [29]. To ensure that the resulting mutant ribosomes contained only the deleted versions of the proteins, a chromosome replacement strategy was used [30].…”

Section: Involvement Of Ribosomal Proteinsmentioning

confidence: 99%

The 30S ribosomal P site: a function of 16S rRNA

Noller¹,

Hoang²,

Fredrick³

2004

FEBS Letters

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The 30S ribosomal P site serves several functions in translation. It must specifically bind initiator tRNA during formation of the 30S initiation complex; bind the anticodon stemloop of peptidyl-tRNA during the elongation phase; and help to maintain the translational reading frame when the A site is unoccupied. Early experiments provided evidence that 16S rRNA was an important component of the 30S P site. Footprinting and crosslinking studies later implicated specific nucleotides in interactions with tRNA. The crystal structures of the 30S subunit and 70S ribosome-tRNA complexes confirmed the interactions between 16S rRNA and tRNA, but also revealed contacts between tRNA and the C-terminal tails of proteins S9 and S13. Deletion of these tails now shows that the 16S rRNA contacts alone are sufficient to support protein synthesis in living cells.

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“…More than one-third of the 54 rproteins possess nonglobular extensions, protruding long distances from globular domains into the RNA-rich core of the ribosome and making extensive contacts with different regions of rRNA Wimberly et al 2000;Brodersen et al 2002). However, the roles of these extensions remain largely unknown and the few that have been studied in some detail, such as the extended loops of Escherichia coli r-proteins S9, S13, L4, and L22, are apparently not required for ribosome assembly and/or function (Zengel et al 2003;Hoang et al 2004). …”

Section: Introductionmentioning

confidence: 99%

The N-terminal extension of Escherichia coli ribosomal protein L20 is important for ribosome assembly, but dispensable for translational feedback control

Guillier

Allemand²,

Graffe³

et al. 2005

RNA

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The Escherichia coli autoregulatory ribosomal protein L20 consists of two structurally distinct domains. The C-terminal domain is globular and sits on the surface of the large ribosomal subunit whereas the N-terminal domain has an extended shape and penetrates deep into the RNA-rich core of the subunit. Many other ribosomal proteins have analogous internal or terminal extensions. However, the biological functions of these extended domains remain obscure. Here we show that the N-terminal tail of L20 is important for ribosome assembly in vivo. Indeed, a truncated version of L20 without its N-terminal tail is unable to complement the deletion of rplT, the gene encoding L20. In addition, this L20 truncation confers a lethal-dominant phenotype, suggesting that the N-terminal domain is essential for cell growth because it could be required for ribosome assembly. Supporting this hypothesis, partial deletions of the N-terminal tail of the protein are shown to cause a slow-growth phenotype due to altered ribosome assembly in vivo as large amounts of intermediate 40S ribosomal particles accumulate. In addition to being a ribosomal protein, L20 also acts as an autogenous repressor. Using L20 truncations, we also show that the N-terminal tail of L20 is dispensable for autogenous control.

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Creating ribosomes with an all-RNA 30S subunit P site

Cited by 74 publications

References 23 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

The 30S ribosomal P site: a function of 16S rRNA

The N-terminal extension of Escherichia coli ribosomal protein L20 is important for ribosome assembly, but dispensable for translational feedback control

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