Ribosome activity and modification of 16S RNA are influenced by deletion of ribosomal protein S20

Rydén-Aulin, Monica; Zhang, Shaoping; Kylsten, Per; Isaksson, Leif A.

doi:10.1111/j.1365-2958.1993.tb01190.x

Cited by 39 publications

(34 citation statements)

References 52 publications

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“…S6B), suggesting that this tertiary contact may stabilize the P4a conformation in full-length 16S rRNA context. The small ribosomal protein S20 can also bind the 126-235 region, and it plays a crucial role in stabilizing this region of the 5 ′ domain (Rydén-Aulin et al 1993;Brodersen et al 2002). These scenarios suggest a novel checkpoint for ribosome assembly based on locking the P4a/ shift-P4a register shift and should be resolvable through future experiments.…”

Section: Excited Statesmentioning

confidence: 99%

High-throughput mutate-map-rescue evaluates SHAPE-directed RNA structure and uncovers excited states

Tian

Cordero

Kladwang

et al. 2014

RNA

101

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The three-dimensional conformations of noncoding RNAs underpin their biochemical functions but have largely eluded experimental characterization. Here, we report that integrating a classic mutation/rescue strategy with high-throughput chemical mapping enables rapid RNA structure inference with unusually strong validation. We revisit a 16S rRNA domain for which SHAPE (selective 2 ′ -hydroxyl acylation with primer extension) and limited mutational analysis suggested a conformational change between apo-and holo-ribosome conformations. Computational support estimates, data from alternative chemical probes, and mutate-and-map (M 2 ) experiments highlight issues of prior methodology and instead give a nearcrystallographic secondary structure. Systematic interrogation of single base pairs via a high-throughput mutation/rescue approach then permits incisive validation and refinement of the M 2 -based secondary structure. The data further uncover the functional conformation as an excited state (20 ± 10% population) accessible via a single-nucleotide register shift. These results correct an erroneous SHAPE inference of a ribosomal conformational change, expose critical limitations of conventional structure mapping methods, and illustrate practical steps for more incisively dissecting RNA dynamic structure landscapes.

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Section: Excited Statesmentioning

confidence: 99%

High-throughput mutate-map-rescue evaluates SHAPE-directed RNA structure and uncovers excited states

Tian

Cordero

Kladwang

et al. 2014

RNA

101

View full text Add to dashboard Cite

show abstract

“…Perhaps there are lesions in the biogenesis cascade that could cause KsgA to bind persistently to the SSU. Interestingly, a mutant lacking r-protein S20 is unable to methylate the substrate of KsgA (Ryden-Aulin et al, 1993), consistent with a role for KsgA as a bona fide maturation check point. The composition of the SSU precursor found in a KsgA E66 mutant could provide vital clues to answer these questions.…”

Section: Discussionmentioning

confidence: 70%

Ribosome biogenesis; the KsgA protein throws a methyl‐mediated switch in ribosome assembly

Mangat

Brown

2008

Molecular Microbiology

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SummaryMany trans-acting factors that aid in ribosome biogenesis have been identified in higher organisms but relatively few such factors are known in prokaryotes. In bacteria, the list of such factors includes ATPenergized helicases and chaperones as well as an emerging cadre of switch GTPases. The KsgA protein is a universally conserved methyltransferase that dimethylates both A1518 and A1519 of the 16S rRNA of the small ribosomal subunit. Methylation has long been thought to be solely for fine-tuning of protein translation. In this issue of Molecular Microbiology, Connolly et al. present data suggesting KsgA might function in the assembly of the small subunit of the ribosome. Indeed, the work indicates that KsgA might have a checkpoint role in ribosome biogenesis where methylation by this protein marks the completion of its assembly role. These findings open our thinking to new candidate assembly factors and provide a new direction for understanding ribosome assembly.Seminal experiments in the 1970s revealed that ribosome assembly could be accomplished in vitro without any assembly factors. Nevertheless, these experiments required lengthy incubations, high temperatures and salt concentrations that are far from physiological (Traub and Nomura, 1969;Nierhaus and Dohme, 1974). Thus, ribosome biogenesis in the cell was thought to be facilitated by extra-ribosomal factors but these have remained elusive in bacteria until relatively recently. These factors include switch GTPases, ATP-dependent RNA helicases and chaperones (Wilson and Nierhaus, 2007). The function of ribosomal methyltransferases was previously thought to be in structural refinement of the ribosome to increase the efficiency of translation. In this issue of Molecular Microbiology, Connolly et al. (2008) have examined the influence of a universally conserved methylase, KsgA, on the maturation of the ribosome and suggest that it might function as a checkpoint during ribosome biogenesis.The ribosome is composed of a large subunit (LSU) and small subunit (SSU) that are produced by a complex and co-ordinated process involving 54 proteins and three rRNAs in bacteria. The rRNA is transcribed as a single 30S co-transcript that is cleaved and processed by specific ribonucleases to produce three rRNAs, 23S and 5S rRNA in the LSU and 16S rRNA in the SSU. During transcription, proteins with direct affinity for the rRNA bind as the transcript is being produced. These primary binding proteins facilitate the binding of secondary r-proteins in a cooperative manner. The process is not believed to proceed in a linear fashion; rather, the assembly map resembles a web of many paths. In addition, base modifications such as methylations and pseudouridylations (isomerization of uracil) of the rRNA and post-translational modifications of r-proteins are important for production of the mature ribosome.A major limitation in understanding the function of the ribosome biogenesis factors is that a substrate for their activity is difficult to isolate and we do not fully understand...

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“…Both types of mutants displayed initiation defects in terms of initiator tRNA binding (Gotz et al, 1990) and subunit association (Gotz et al, 1989;Ryden-Aulin et al, 1993). In another study, an S20 knockout mutant was obtained as a suppressor of erythromycin dependence and produced precursor 30S particles and fewer 70S complexes, as measured by density ultracentrifugation (Wild, 1988).…”

Section: Phenotype Of S20 Deletion Mutantmentioning

confidence: 99%

“…Knockout mutants of S20 have been isolated previously as antibiotic dependent revertants and as a suppressor of nonsense codons (Dabbs, 1978;Dabbs, 1979;Ryden-Aulin et al, 1993). Both types of mutants displayed initiation defects in terms of initiator tRNA binding (Gotz et al, 1990) and subunit association (Gotz et al, 1989;Ryden-Aulin et al, 1993).…”

Section: Phenotype Of S20 Deletion Mutantmentioning

confidence: 99%

Comprehending a Molecular Conundrum: Functional Studies of Ribosomal Protein Mutants from Salmonella enterica Serovar Typhimurium

Kåhrström¹,

Andersson²,

Sanyal³

2012

Salmonella - Distribution, Adaptation, Control Measures and Molecular Technologies

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Ribosome activity and modification of 16S RNA are influenced by deletion of ribosomal protein S20

Cited by 39 publications

References 52 publications

High-throughput mutate-map-rescue evaluates SHAPE-directed RNA structure and uncovers excited states

High-throughput mutate-map-rescue evaluates SHAPE-directed RNA structure and uncovers excited states

Ribosome biogenesis; the KsgA protein throws a methyl‐mediated switch in ribosome assembly

Comprehending a Molecular Conundrum: Functional Studies of Ribosomal Protein Mutants from Salmonella enterica Serovar Typhimurium

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