An assembly landscape for the 30S ribosomal subunit

Talkington, Megan W. T.; Siuzdak, Gary; Williamson, James R.

doi:10.1038/nature04261

Cited by 241 publications

(342 citation statements)

References 35 publications

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“…The Nomura maps documented how the omission of one protein precludes incorporation of others, but only monitored stable protein-RNA interactions that would survive the biochemical purification steps including nonequilibrium ultracentrifugation. Recent RNA protection data suggest that weak protein-RNA interactions form rapidly during assembly (5,6), raising the question of how completely the map represents the types of intermediates that form during assembly.…”

mentioning

confidence: 99%

“…Approximate rate constants and orders of binding are known from kinetic experiments, but kinetic profiles for individual protein binding and RNA folding steps are complex and defy simple models (1)(2)(3)(4)(5)(6)(7)(8). The fundamentally kinetic process of ribosome assembly is thus still best understood in terms of the thermodynamic (9) maps generated by the Nomura group in the 1970s (10).…”

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confidence: 99%

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Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy

Ridgeway

Millar²,

Williamson

2012

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

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The self-assembly of bacterial 30S ribosomes involves a large number of RNA folding and RNA-protein binding steps. The sequence of steps determines the overall assembly mechanism and the structure of the mechanism has ramifications for the robustness of biogenesis and resilience against kinetic traps. Thermodynamic interdependencies of protein binding inferred from omission-reconstitution experiments are thought to preclude certain assembly pathways and thus enforce ordered assembly, but this concept is at odds with kinetic data suggesting a more parallel assembly landscape. A major challenge is deconvolution of the statistical distribution of intermediates that are populated during assembly at high concentrations approaching in vivo assembly conditions. To specifically resolve the intermediates formed by binding of three ribosomal proteins to the full length 16S rRNA, we introduce Fluorescence Triple-Correlation Spectroscopy (F3CS). F3CS identifies specific ternary complexes by detecting coincident fluctuations in three-color fluorescence data. Triple correlation integrals quantify concentrations and diffusion kinetics of triply labeled species, and F3CS data can be fit alongside auto-correlation and cross-correlation data to quantify the populations of 10 specific ribosome assembly intermediates. The distribution of intermediates generated by binding three ribosomal proteins to the entire native 16S rRNA included significant populations of species that were not previously thought to be thermodynamically accessible, questioning the current interpretation of the classic omission-reconstitution experiments. F3CS is a general approach for analyzing assembly and function of macromolecular complexes, especially those too large for traditional biophysical methods.fluorescence cross-correlation spectroscopy | fluorescence correlation spectroscopy | RNA-protein interactions T he complete assembly mechanism for the 30S ribosome has remained elusive. Approximate rate constants and orders of binding are known from kinetic experiments, but kinetic profiles for individual protein binding and RNA folding steps are complex and defy simple models (1-8). The fundamentally kinetic process of ribosome assembly is thus still best understood in terms of the thermodynamic (9) maps generated by the Nomura group in the 1970s (10). The Nomura maps documented how the omission of one protein precludes incorporation of others, but only monitored stable protein-RNA interactions that would survive the biochemical purification steps including nonequilibrium ultracentrifugation. Recent RNA protection data suggest that weak protein-RNA interactions form rapidly during assembly (5, 6), raising the question of how completely the map represents the types of intermediates that form during assembly.Nomura dependencies exist between three specific proteins, S7, S9, and S19, that bind the 16S rRNA (Fig. 1A) late in assembly (2, 4-8). In principle, binding of these three proteins to RNA could occur by any and all possible parallel pathways that are il...

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mentioning

confidence: 99%

mentioning

confidence: 99%

Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy

Ridgeway

Millar²,

Williamson

2012

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

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“…15 N-labeled proteins were incubated with 16S rRNA and, after assembly had taken place for various times, chased with an excess of 14 N-labeled proteins. 30S subunits were then completely assembled and purifi ed, and the 15 N/ 14 N ratio in their proteins was used to reveal the binding kinetics [ 34 ]. In this chapter we describe the quantitative analysis of the dynamic protein changes that occur during premRNA splicing by using stable isotope labeling and subsequent mass spectrometry.…”

Section: Eukaryotic Pre-mrnasmentioning

confidence: 99%

Analyzing the Protein Assembly and Dynamics of the Human Spliceosome with SILAC

Schmidt

Raabe

Lührmann

et al. 2014

Methods in Molecular Biology

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Quantitative mass spectrometry has become an indispensable tool in proteomic studies. Numerous methods are available and can be applied to approach different issues. In most studies these issues include the quantitative comparison of different cell states, the identifi cation of specifi c interaction partners or determining degrees of posttranslational modifi cation. In this chapter we describe a SILAC-based quantifi cation in order to analyze dynamic protein changes during the assembly of the human spliceosome on a pre-mRNA in vitro. We provide protocols for assembly of spliceosomes on pre-mRNA (including generation of pre-mRNAs and preparation of nuclear extracts), quantitative mass spectrometry (SILAC labeling, sample preparation), and data analysis to generate timelines for the dynamic protein assembly.

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“…24 In the course of extending the ribosome assembly work to metabolic labeling in rapidly growing E. coli, we encountered complex isotope distributions due to fractional 15 N-labeling of peptides derived from ribosomal proteins. Consider the experimentally observed peaks shown in Figure 1, the mass spectrum of a peptide from bacterial cells that were treated with a pulse of 15 N-ammonium sulfate for 100 min prior to harvesting the cells.…”

Section: Analytical Description Of a Composite Unlabeled And Fractionmentioning

confidence: 99%

“…A residue composition vector r̂j can be defined in analogy to n, whose elements give the atomic composition of that residue. The complete set of residue composition vectors can be assembled into a residue composition matrix: (24) The residue formula is given by a vector p̂ of length n residues , where the elements specify the number of each residue type in the species. The relationship between the molecular formula and residue formula is given by: (25) The μ-domain representation f(μ) can then be represented in the alternative residue basis (26) where (27) Essentially the expressions in eqs 26 and 27 factor the μ-domain function in eq 4 according to residues instead of atoms.…”

Section: Residue-based Convolution Of Isotope Distributionsmentioning

confidence: 99%

Quantitative Analysis of Isotope Distributions In Proteomic Mass Spectrometry Using Least-Squares Fourier Transform Convolution

et al. 2008

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Quantitative proteomic mass spectrometry involves comparison of the amplitudes of peaks resulting from different isotope labeling patterns, including fractional atomic labeling and fractional residue labeling. We have developed a general and flexible analytical treatment of the complex isotope distributions that arise in these experiments, using Fourier transform convolution to calculate labeled isotope distributions and least-squares for quantitative comparison with experimental peaks. The degree of fractional atomic and fractional residue labeling can be determined from experimental peaks at the same time as the integrated intensity of all of the isotopomers in the isotope distribution. The approach is illustrated using data with fractional 15 N-labeling and fractional 13 C-isoleucine labeling. The least-squares Fourier transform convolution approach can be applied to many types of quantitive proteomic data, including data from stable isotope labeling by amino acids in cell culture and pulse labeling experiments.Stable isotope labeling in cells coupled with mass spectrometry has many important applications in the analysis of protein expression, modification, turnover, and metabolism. 1 Cells and organisms can be isotopically labeled by supplying labeled precursors in the form of nutrients such as amino acids, glucose, and ammonia. These labeled precursors are incorporated into proteins, whose resulting isotope labeling patterns reflect their abundance and the dynamics of protein synthesis and turnover. The era of quantitative proteomics began with experiments where mixtures of unlabeled control cells and 15 N-labeled or isotope depleted test cells were quantitatively analyzed to determine protein expression and phosphorylation levels. 2,3 More recently, the SILAC technique was developed based on specific amino acid labeling of cells for quantitative analysis of protein expression, modification, and turnover. 4-6The two basic classes of quantitative metabolic labeling approaches are the stable isotope labeling by amino acids in cell culture (SILAC) experiments and pulse labeling experiments, both of which have been implemented in a variety of ways. 7 In the SILAC method, independent samples are prepared with different labeling patterns that are mixed prior to mass spectrometry analysis. For pulse labeling experiments, labels that are added to the growth medium are taken up to metabolically label the proteome, which generates fractionally enriched proteins from a

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An assembly landscape for the 30S ribosomal subunit

Cited by 241 publications

References 35 publications

Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy

Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy

Analyzing the Protein Assembly and Dynamics of the Human Spliceosome with SILAC

Quantitative Analysis of Isotope Distributions In Proteomic Mass Spectrometry Using Least-Squares Fourier Transform Convolution

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