The DnaK Chaperone System Facilitates 30S Ribosomal Subunit Assembly

Maki, Jennifer A.; Schnobrich, Daniel J; Culver, Gloria M.

doi:10.1016/s1097-2765(02)00562-2

Cited by 90 publications

(92 citation statements)

References 42 publications

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“…Cells were grown at 37°C (permissive temperature) to ∼0.7 OD or grown at 37°C to ∼0.2 OD prior to being shifted to 20°C, which is the nonpermissive temperature for S5 (G28D) cells or room temperature for ΔrpsO cells and incubated for another 4 h. RimJ expression in S5(G28D) strain was done as described earlier (11). Ribosome profiles were essentially performed as described (37), with sedimentation adjusted to allow separation of SSUs and pre-SSUs at the expense of observing polysomes. RNA extraction and primer extension were performed as described (38).…”

Section: Discussionmentioning

confidence: 99%

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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Section: Discussionmentioning

confidence: 99%

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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“…G2 regions contain a conserved threonine, but otherwise differ between GTPases of different subfamilies and are responsible for coordination of a Mg 21 ion that binds to the b-and c-phosphates. Because in ras-like GTPases, this region shows often large structural differences between the GTP and GDP bound states, it is also referred to as the switch I region.…”

Section: G1 [Gxxxxgk(s/t)]mentioning

confidence: 99%

“…The intermediate observed upon YihA depletion has a slightly slower sedimentation rate than the 45S intermediates observed when RbgA and EngA are depleted. [46][47][48] In contrast, depletion of Obg leads to accumulation of a 50S pre-ribosome that is unstable at low Mg 21 concentrations. 49 The increasing sedimentation rate observed with intermediates accumulating when YihA, RbgA, EngA, and Obg are depleted, suggest a preliminary order to the action of these GTPases with YihA acting before RbgA and EngA, and Obg being the last GTPase to act on assembling 50S subunits ( Figure 4B).…”

Section: Gtpases Constitute a Large And Diverse Class Of Ribosome Assmentioning

confidence: 99%

“…RsgA contains an OB fold as found in the ribosomal protein S1. Both RsgA and YqeH contain Zn 21 -binding loops that are likely RNA binding elements. 52,53 Obg has glycine-rich N-terminal domain found in all Obg proteins 54 as well as a less conserved TGS domain also found in threonine tRNA synthetases, GTPases, and SpoT.…”

Section: 9mentioning

confidence: 99%

“…For example, it was shown that the DnaK chaperone system promoted a conformational rearrangement in assembling ribosomes, which otherwise requires a heating step. 21 Furthermore, a combination of genetic, genomic, and biochemical work has revealed that assembly of bacterial ribosomes in vivo was promoted by the action of several accessory proteins, some of which are essential (e.g., Refs. [15][16][17][18][19]22,23).…”

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confidence: 99%

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Role of GTPases in ribosome assembly

Karbstein

2007

Biopolymers

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GTPases are a universally conserved class of regulatory proteins involved in such diverse cellular functions as signal transduction, translation, cytoskeleton formation, and intracellular transport. GTPases are also required for ribosome assembly in eukaryotes and bacteria, where they present themselves as possible regulatory molecules. Strikingly, in bacteria they represent the largest class of essential assembly factors. A review of their common structural, biochemical and genetic interactions is presented and integrated with models for their function in ribosome assembly. © 2007 Wiley Periodicals, Inc. Biopolymers 87: 1–11, 2007This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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Factor‐Mediated Ribosome Assembly in Bacteria

Dönhöfer¹,

Sharma

et al. 2009

Encyclopedia of Life Sciences

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The ribosome is a structurally and functionally conserved macromolecular machine responsible for translating messenger ribonucleic acid (mRNA) into proteins. Composed of two independently assembled subunits, the bacterial ribosome is approximately 2.5 MDa in size. The process of translation, catalysed by the ribosome, is central to gene expression in the cell. Correspondingly, the regulation of ribosome biogenesis is paramount to cell viability, growth and differentiation. Moreover, the ribosome is the target of numerous clinically relevant therapeutic agents, several of which are known to affect ribosome assembly. Although, in vitro reconstitution of bacterial ribosomal subunits can be achieved using purified ribosomal RNA (rRNA) and r‐protein components, the process occurs at nonphysiological conditions. It is suggested that a specific order of processing and assembly events and regulatory factors is required to achieve fully functional particles. This article focuses on the protein factors that are involved in the process of bacterial ribosome biogenesis. Key concepts: This article focuses on the protein factors that are involved in the process of bacterial ribosome biogenesis.

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The DnaK Chaperone System Facilitates 30S Ribosomal Subunit Assembly

Cited by 90 publications

References 42 publications

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

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

Role of GTPases in ribosome assembly

Factor‐Mediated Ribosome Assembly in Bacteria

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