Formation of the central pseudoknot in 16S rRNA is essential for initiation of translation.

Brink, Marcel F.; Verbeet, Martin Ph.; Boer, H. H.

doi:10.1002/j.1460-2075.1993.tb06076.x

Cited by 48 publications

(55 citation statements)

References 34 publications

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“…Previous studies have shown that mutation of the residue G917 significantly perturbs the translational activity of ribosomes (Brink et al 1993). This is consistent with our results using the specialized ribosome system (Fig.…”

Section: Discussionsupporting

confidence: 93%

Differential assembly of 16S rRNA domains during 30S subunit formation

Xu¹,

Culver²

2010

RNA

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Rapid and accurate assembly of the ribosomal subunits, which are responsible for protein synthesis, is required to sustain cell growth. Our best understanding of the interaction of 30S ribosomal subunit components (16S ribosomal RNA [rRNA] and 20 ribosomal proteins [r-proteins]) comes from in vitro work using Escherichia coli ribosomal components. However, detailed information regarding the essential elements involved in the assembly of 30S subunits still remains elusive. Here, we defined a set of rRNA nucleotides that are critical for the assembly of the small ribosomal subunit in E. coli. Using an RNA modification interference approach, we identified 54 nucleotides in 16S rRNA whose modification prevents the formation of a functional small ribosomal subunit. The majority of these nucleotides are located in the head and interdomain junction of the 30S subunit, suggesting that these regions are critical for small subunit assembly. In vivo analysis of specific identified sites, using engineered mutations in 16S rRNA, revealed defective protein synthesis capability, aberrant polysome profiles, and abnormal 16S rRNA processing, indicating the importance of these residues in vivo. These studies reveal that specific segments of 16S rRNA are more critical for small subunit assembly than others, and suggest a hierarchy of importance.

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Section: Discussionsupporting

confidence: 93%

Differential assembly of 16S rRNA domains during 30S subunit formation

Xu¹,

Culver²

2010

RNA

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“…Furthermore, it has been proposed that box C sequences, part of the nut-like elements and harboring the RNase III cleavage site, might mediate the formation of the universally conserved central pseudoknot of the 30S subunit (24). Mutational studies in the pseudoknot region revealed that the mutant ribosomes were impaired for in vivo translation (15,33), establishing the significance of the central pseudoknot to ribosome function. Formation of the universally conserved pseudoknot is believed to be preceded by another pseudoknot structure that involves base pairing of a U3 box A-like sequence between positions −104 and −122 of the 5 0 leader of 16S rRNA and NTs 14-31 and 916-918 of 16S rRNA (24).…”

Section: Discussionmentioning

confidence: 97%

Appropriate maturation and folding of 16S rRNA during 30S subunit biogenesis are critical for translational fidelity

Roy-Chaudhuri

Kirthi

Culver

2010

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

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Ribosomal protein S5 is critical for small ribosomal subunit (SSU) assembly and is indispensable for SSU function. Previously, we identified a point mutation in S5, (G28D) that alters both SSU formation and translational fidelity in vivo, which is unprecedented for other characterized S5 mutations. Surprisingly, additional copies of an extraribosomal assembly factor, RimJ, rescued all the phenotypes associated with S5(G28D), including fidelity defects, suggesting that the effect of RimJ on rescuing the miscoding of S5(G28D) is indirect. To understand the underlying mechanism, we focused on the biogenesis cascade and observed defects in processing of precursor 16S (p16S) rRNA in the S5(G28D) strain, which were rescued by RimJ. Analyses of p16S rRNA-containing ribosomes from other strains further supported a correspondence between the extent of 5 0 end maturation of 16S rRNA and translational miscoding. Chemical probing of mutant ribosomes with additional leader sequences at the 5 0 end of 16S rRNA compared to WT ribosomes revealed structural differences in the region of helix 1. Thus, the presence of additional nucleotides at the 5 0 end of 16S rRNA could alter fidelity by changing the architecture of 16S rRNA in translating ribosomes and suggests that fidelity is governed by accuracy and completeness of the SSU biogenesis cascade.R-protein S5 | ram mutations | ribosome biogenesis | 16S rRNA processing O ne of the most remarkable feats of the ribosome is the ability to decode genetic information accurately in a process that involves the interaction of aminoacyl-transfer RNAs (aa-tRNAs) to the aa-tRNA binding site (A site) on the small ribosomal subunit (SSU; 30S). However, this process is not fully error proof and missense errors occur at a frequency of 10 −3 to 10 −4 per amino acid synthesized (1). Decoding is thought to involve a number of local and global conformational changes in the SSU upon binding of a cognate aa-tRNA to the A site. These structural changes result in a transition from the "open" to the "closed" form whereby the head of the SSU rotates toward the shoulder and the shoulder toward the platform (2). The r-proteins S4, S5, and S12 along with helices 27 and 44 of 16S rRNA are implicated in fidelity; mutations in S4 and S5 can lead to ribosome ambiguity (ram) or miscoding, whereas specific mutations in S12 lead to hyperaccuracy (3). Based on the recent crystal structures (2), the r-protein ram mutations map at the interface of S4 and S5 and disrupt a number of salt bridges that are present in the open SSUs. These changes could destabilize the open state, thereby perturbing the equilibrium to promote the closed state and allowing decreased discrimination in the decoding process (4). Some additional biochemical and structural data support this model (5); nonetheless, other data are hard to incorporate into this scheme. A few mutations in S4 can confer "restrictive" phenotypes to Salmonella typhimurium (6) and surprisingly these hyperaccurate alleles of S4 suppress the hyperaccurate phenotypes of S1...

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“…At the other extremity of helix 27, the adjacent segment encompassing nucleotides 913-920 interacts with the loop of helix 1, forming helix 2, as described above, which creates the central pseudoknot of the 30S subunit. Mutations that disrupt the central pseudoknot destabilize the 30S subunit and impair subunit association, demonstrating the importance of the central pseudoknot for ribosome function (Brink et al 1993;Poot et al 1996). It had been suggested that the central pseudoknot is subject to conformational rearrangements involving alternate base pairings during protein synthesis (Kössel et al 1990;Leclerc and BrakierGingras 1991), but these rearrangements were not supported by mutagenesis experiments (Pinard et al 1993;Poot et al 1998).…”

Section: Discussionmentioning

confidence: 99%

A functional relationship between helix 1 and the 900 tetraloop of 16S ribosomal RNA within the bacterial ribosome

Bart

Théberge-Julien

Cunningham³

et al. 2005

RNA

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The conserved 900 tetraloop that caps helix 27 of 16S ribosomal RNA (rRNA) interacts with helix 24 of 16S rRNA and also with helix 67 of 23S rRNA, forming the intersubunit bridge B2c, proximal to the decoding center. In previous studies, we investigated how the interaction between the 900 tetraloop and helix 24 participates in subunit association and translational fidelity. In the present study, we investigated whether the 900 tetraloop is involved in other undetected interactions with different regions of the Escherichia coli 16S rRNA. Using a genetic complementation approach, we selected mutations in 16S rRNA that compensate for a 900 tetraloop mutation, A900G, which severely impairs subunit association and translational fidelity. Mutations were randomly introduced in 16S rRNA, using either a mutagenic XL1-Red E. coli strain or an error-prone PCR strategy. Gain-offunction mutations were selected in vivo with a specialized ribosome system. Two mutations, the deletion of U12 and the U12C substitution, were thus independently selected in helix 1 of 16S rRNA. This helix is located in the vicinity of helix 27, but does not directly contact the 900 tetraloop in the crystal structures of the ribosome. Both mutations correct the subunit association and translational fidelity defects caused by the A900G mutation, revealing an unanticipated functional interaction between these two regions of 16S rRNA.

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Formation of the central pseudoknot in 16S rRNA is essential for initiation of translation.

Cited by 48 publications

References 34 publications

Differential assembly of 16S rRNA domains during 30S subunit formation

Differential assembly of 16S rRNA domains during 30S subunit formation

Appropriate maturation and folding of 16S rRNA during 30S subunit biogenesis are critical for translational fidelity

A functional relationship between helix 1 and the 900 tetraloop of 16S ribosomal RNA within the bacterial ribosome

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