In Vivo X-Ray Footprinting of Pre-30S Ribosomes Reveals Chaperone-Dependent Remodeling of Late Assembly Intermediates

Soper, Sarah F. Clatterbuck; Dator, Romel P; Limbach, Patrick A.; Woodson, Sarah A.

doi:10.1016/j.molcel.2013.09.020

Cited by 96 publications

(120 citation statements)

References 61 publications

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“…The uS5 protein is a tertiary binding protein and is essential for correct assembly of the 30S subunit (27,28). When lysates from each of the 21 mutants (grown at 37°C) were analyzed by sucrose gradient centrifugation, 6 mutants (V20E, G27C, G27W, ΔV24/G27S, K25N/G27N, and ΔK25/G26S/G27S) showed substantially increased levels of free 50S and 30S subunits.…”

Section: Resultsmentioning

confidence: 99%

The Loop 2 Region of Ribosomal Protein uS5 Influences Spectinomycin Sensitivity, Translational Fidelity, and Ribosome Biogenesis

Kamath

Gregory

O’Connor

2017

Antimicrob Agents Chemother

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Ribosomal protein uS5 is an essential component of the small ribosomal subunit that is involved in subunit assembly, maintenance of translational fidelity, and the ribosome's response to the antibiotic spectinomycin. While many of the characterized uS5 mutations that affect decoding map to its interface with uS4, more recent work has shown that residues distant from the uS4-uS5 interface can also affect the decoding process. We targeted one such interface-remote area, the loop 2 region (residues 20 to 31), for mutagenesis in Escherichia. coli and generated 21 unique mutants. A majority of the loop 2 alterations confer resistance to spectinomycin and affect the fidelity of translation. However, only a minority show altered rRNA processing or ribosome biogenesis defects.

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Section: Resultsmentioning

confidence: 99%

The Loop 2 Region of Ribosomal Protein uS5 Influences Spectinomycin Sensitivity, Translational Fidelity, and Ribosome Biogenesis

Kamath

Gregory

O’Connor

2017

Antimicrob Agents Chemother

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“…Third, both the beak structure and the platform region are initially misfolded and then refolded during the heat-dependent activation step (95). Finally, and intriguingly, just as Ltv1 modulates the incorporation of S10 and S3 in eukaryotes (57), RimM modulates the assembly of the bacterial ortholog of S3 (also known as S3), by regulating folding of 16S rRNA (96). Other examples of an assembly factor delaying r-protein incorporation are discussed in the section titled Roles of R-Protein Paralogs: The Placeholder Hypothesis, below.…”

Section: Modulation Of 40s R-protein Binding By Assembly Factorsmentioning

confidence: 99%

Functions of Ribosomal Proteins in Assembly of Eukaryotic Ribosomes In Vivo

Cruz

Karbstein

Woolford

2015

Annu. Rev. Biochem.

323

241

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The proteome of cells is synthesized by ribosomes, complex ribonucleoproteins that in eukaryotes contain 79–80 proteins and four ribosomal RNAs (rRNAs) more than 5,400 nucleotides long. How these molecules assemble together and how their assembly is regulated in concert with the growth and proliferation of cells remain important unanswered questions. Here, we review recently emerging principles to understand how eukaryotic ribosomal proteins drive ribosome assembly in vivo. Most ribosomal proteins assemble with rRNA cotranscriptionally; their association with nascent particles is strengthened as assembly proceeds. Each subunit is assembled hierarchically by sequential stabilization of their subdomains. The active sites of both subunits are constructed last, perhaps to prevent premature engagement of immature ribosomes with active subunits. Late-assembly intermediates undergo quality-control checks for proper function. Mutations in ribosomal proteins that affect mostly late steps lead to ribosomopathies, diseases that include a spectrum of cell type–specific disorders that often transition from hypoproliferative to hyperproliferative growth.

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“…The method has since successfully tackled ambitious complex targets such as the acto-myosin complex 53 . Unlike most of the other structural biology methods XF-MS is not limited by the size of the studied complex, and as such it is very well suited for analysis of multiprotein complexes, including megadalton assemblies such as the Clp proteasome 54 and even ribosome assemblies 55,56 . While these ribosomal works are following the RNA-patterns, and are not strictly speaking dealing with protein-protein interactions, the latter is particularly significant as it describes an in vivo application of XRF.…”

Section: Complexesmentioning

confidence: 99%

Oxidative footprinting in the study of structure and function of membrane proteins: current state and perspectives

Bavro

Gupta

Ralston

2015

Biochemical Society Transactions

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Abstract:Membrane proteins, such as receptors, transporters and ion channels control the vast majority of cellular signaling and metabolite exchange processes and as such are increasingly becoming the key pharmacological targets. Difficulties of handling membrane proteins in high concentrations and requirements of membrane environments for their stabilization have made them difficult to study using traditional structural biology techniques, requiring the use of a hybrid, integrative approaches to study their dynamic properties and functional aspects in physiologically relevant conditions. In recent years significant progress has been made in the field of oxidative labeling techniques, and particularly X-radiolytic labeling in combination of mass spectroscopy (XF-MS) allowing these approaches to mature and provide valuable insights into structure and function of proteins, including membrane targets, which is difficult to obtain by other techniques, complementing available structural data. XF-MS has demonstrated unique capability for identification of structural waters and conformational changes in proteins at both high degree of spatial and temporal resolution. Here we provide a perspective of the place of XF-MS amongst other structural biology methods and showcase some of latest developments in usage of XF-MS in solving water meditated transmembrane signaling, ion transport, ion gating as well as ligand induced allosteric conformation changes in these membrane protein complexes.

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In Vivo X-Ray Footprinting of Pre-30S Ribosomes Reveals Chaperone-Dependent Remodeling of Late Assembly Intermediates

Cited by 96 publications

References 61 publications

The Loop 2 Region of Ribosomal Protein uS5 Influences Spectinomycin Sensitivity, Translational Fidelity, and Ribosome Biogenesis

The Loop 2 Region of Ribosomal Protein uS5 Influences Spectinomycin Sensitivity, Translational Fidelity, and Ribosome Biogenesis

Functions of Ribosomal Proteins in Assembly of Eukaryotic Ribosomes In Vivo

Oxidative footprinting in the study of structure and function of membrane proteins: current state and perspectives

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