Michael T Sykes

The Scripps Research Institute, لا هویا, California, United States

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Publications (7)73.01 Total impact

  • Zahra Shajani · Michael T Sykes · James R Williamson ·
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    ABSTRACT: The assembly of ribosomes from a discrete set of components is a key aspect of the highly coordinated process of ribosome biogenesis. In this review, we present a brief history of the early work on ribosome assembly in Escherichia coli, including a description of in vivo and in vitro intermediates. The assembly process is believed to progress through an alternating series of RNA conformational changes and protein-binding events; we explore the effects of ribosomal proteins in driving these events. Ribosome assembly in vivo proceeds much faster than in vitro, and we outline the contributions of several of the assembly cofactors involved, including Era, RbfA, RimJ, RimM, RimP, and RsgA, which associate with the 30S subunit, and CsdA, DbpA, Der, and SrmB, which associate with the 50S subunit.
    Annual review of biochemistry 06/2011; 80(1):501-26. DOI:10.1146/annurev-biochem-062608-160432 · 30.28 Impact Factor
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    ABSTRACT: Although high-resolution structures of the ribosome have been solved in a series of functional states, relatively little is known about how the ribosome assembles, particularly in vivo. Here, a general method is presented for studying the dynamics of ribosome assembly and ribosomal assembly intermediates. Since significant quantities of assembly intermediates are not present under normal growth conditions, the antibiotic neomycin is used to perturb wild-type Escherichia coli. Treatment of E. coli with the antibiotic neomycin results in the accumulation of a continuum of assembly intermediates for both the 30S and 50S subunits. The protein composition and the protein stoichiometry of these intermediates were determined by quantitative mass spectrometry using purified unlabeled and (15)N-labeled wild-type ribosomes as external standards. The intermediates throughout the continuum are heterogeneous and are largely depleted of late-binding proteins. Pulse-labeling with (15)N-labeled medium time-stamps the ribosomal proteins based on their time of synthesis. The assembly intermediates contain both newly synthesized proteins and proteins that originated in previously synthesized intact subunits. This observation requires either a significant amount of ribosome degradation or the exchange or reuse of ribosomal proteins. These specific methods can be applied to any system where ribosomal assembly intermediates accumulate, including strains with deletions or mutations of assembly factors. This general approach can be applied to study the dynamics of assembly and turnover of other macromolecular complexes that can be isolated from cells.
    Journal of Molecular Biology 10/2010; 403(3):331-45. DOI:10.1016/j.jmb.2010.08.005 · 4.33 Impact Factor
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    Michael T Sykes · Edit Sperling · Stephen S Chen · James R Williamson ·
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    ABSTRACT: Relative levels of ribosomal proteins were quantified in crude cell lysates using mass spectrometry. A method for quantifying cellular protein levels using macromolecular standards is presented that does not require complex sample separation, identification of high-responding peptides, affinity purification, or postgrowth modifications. Perturbations in ribosomal protein levels by overexpression of individual proteins correlate to known autoregulatory mechanisms and extend the network of ribosomal protein regulation.
    Analytical Chemistry 06/2010; 82(12):5038-45. DOI:10.1021/ac9028664 · 5.64 Impact Factor
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    ABSTRACT: Escherichia coli DbpA is an ATP-dependent RNA helicase with specificity for hairpin 92 of 23S ribosomal RNA, an important part of the peptidyl transferase center. The R331A active site mutant of DbpA confers a dominant slow growth and cold sensitive phenotype when overexpressed in E. coli containing endogenous DbpA. Ribosome profiles from cells overexpressing DbpA R331A display increased levels of 50S and 30S subunits and decreased levels 70S ribosomes. Profiles run at low Mg2+ exhibit fewer 50S subunits and accumulate a 45S particle that contains incompletely processed and undermodified 23S rRNA in addition to reduced levels of several ribosomal proteins that bind late in the assembly pathway. Unlike mature 50S subunits, these 45S particles can stimulate the ATPase activity of DbpA, indicating that hairpin 92 has not yet been sequestered within the 50S subunit. Overexpression of the inactive DbpA R331A mutant appears to block assembly at a late stage when the peptidyl transferase center is formed, indicating a possible role for DbpA promoting this conformational change.
    Nucleic Acids Research 10/2009; 37(19):6503-14. DOI:10.1093/nar/gkp711 · 9.11 Impact Factor
  • Michael T Sykes · James R Williamson ·
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    ABSTRACT: The ribosome is a complex macromolecular machine responsible for protein synthesis in the cell. It consists of two subunits, each of which contains both RNA and protein components. Ribosome assembly is subject to intricate regulatory control and is aided by a multitude of assembly factors in vivo, but can also be carried out in vitro. The details of the assembly process remain unknown even in the face of atomic structures of the entire ribosome and after more than three decades of research. Some of the earliest research on ribosome assembly produced the Nomura assembly map of the small subunit, revealing a hierarchy of protein binding dependencies for the 20 proteins involved and suggesting the possibility of a single intermediate. Recent work using a combination of RNA footprinting and pulse-chase quantitative mass spectrometry paints a picture of small subunit assembly as a dynamic and varied landscape, with sequential and hierarchical RNA folding and protein binding events finally converging on complete subunits. Proteins generally lock tightly into place in a 5' to 3' direction along the ribosomal RNA, stabilizing transient RNA conformations, while RNA folding and the early stages of protein binding are initiated from multiple locations along the length of the RNA.
    Annual Review of Biophysics 02/2009; 38(1):197-215. DOI:10.1146/annurev.biophys.050708.133615 · 15.44 Impact Factor
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    Michael T Sykes · James R Williamson ·
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    ABSTRACT: An important aspect of proteomic mass spectrometry involves quantifying and interpreting the isotope distributions arising from mixtures of macromolecules with different isotope labeling patterns. These patterns can be quite complex, in particular with in vivo metabolic labeling experiments producing fractional atomic labeling or fractional residue labeling of peptides or other macromolecules. In general, it can be difficult to distinguish the contributions of species with different labeling patterns to an experimental spectrum and difficult to calculate a theoretical isotope distribution to fit such data. There is a need for interactive and user-friendly software that can calculate and fit the entire isotope distribution of a complex mixture while comparing these calculations with experimental data and extracting the contributions from the differently labeled species. Envelope has been developed to be user-friendly while still being as flexible and powerful as possible. Envelope can simultaneously calculate the isotope distributions for any number of different labeling patterns for a given peptide or oligonucleotide, while automatically summing these into a single overall isotope distribution. Envelope can handle fractional or complete atom or residue-based labeling, and the contribution from each different user-defined labeling pattern is clearly illustrated in the interactive display and is individually adjustable. At present, Envelope supports labeling with 2H, 13C, and 15N, and supports adjustments for baseline correction, an instrument accuracy offset in the m/z domain, and peak width. Furthermore, Envelope can display experimental data superimposed on calculated isotope distributions, and calculate a least-squares goodness of fit between the two. All of this information is displayed on the screen in a single graphical user interface. Envelope supports high-quality output of experimental and calculated distributions in PNG or PDF format. Beyond simply comparing calculated distributions to experimental data, Envelope is useful for planning or designing metabolic labeling experiments, by visualizing hypothetical isotope distributions in order to evaluate the feasibility of a labeling strategy. Envelope is also useful as a teaching tool, with its real-time display capabilities providing a straightforward way to illustrate the key variable factors that contribute to an observed isotope distribution. Envelope is a powerful tool for the interactive calculation and visualization of complex isotope distributions for comparison to experimental data. It is available under the GNU General Public License from http://williamson.scripps.edu/envelope/.
    BMC Bioinformatics 11/2008; 9(1):446. DOI:10.1186/1471-2105-9-446 · 2.58 Impact Factor
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    Edit Sperling · Anne E Bunner · Michael T Sykes · James R Williamson ·
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    ABSTRACT: 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 quantitative proteomic data, including data from stable isotope labeling by amino acids in cell culture and pulse labeling experiments.
    Analytical Chemistry 07/2008; 80(13):4906-17. DOI:10.1021/ac800080v · 5.64 Impact Factor

Publication Stats

302 Citations
73.01 Total Impact Points


  • 2008-2011
    • The Scripps Research Institute
      • Skaggs Institute for Chemical Biology
      لا هویا, California, United States
  • 2009
    • Northwestern University
      Evanston, Illinois, United States