23 research outputs found

    Quantitative Analysis of rRNA Modifications Using Stable Isotope Labeling and Mass Spectrometry

    No full text
    Post-transcriptional RNA modifications that are introduced during the multistep ribosome biogenesis process are essential for protein synthesis. The current lack of a comprehensive method for a fast quantitative analysis of rRNA modifications significantly limits our understanding of how individual modification steps are coordinated during biogenesis inside the cell. Here, an LC-MS approach has been developed and successfully applied for quantitative monitoring of 29 out of 36 modified residues in the 16S and 23S rRNA from Escherichia coli. An isotope labeling strategy is described for efficient identification of ribose and base methylations, and a novel metabolic labeling approach is presented to allow identification of MS-silent pseudouridine modifications. The method was used to measure relative abundances of modified residues in incomplete ribosomal subunits compared to a mature <sup>15</sup>N-labeled rRNA standard, and a number of modifications in both 16S and 23S rRNA were present in substoichiometric amounts in the preribosomal particles. The RNA modification levels correlate well with previously obtained profiles for the ribosomal proteins, suggesting that RNA is modified in a schedule comparable to the association of the ribosomal proteins. Importantly, this study establishes an efficient workflow for a global monitoring of ribosomal modifications that will contribute to a better understanding of mechanisms of RNA modifications and their impact on intracellular processes in the future

    Supplementary Figure 1 from Structure and dynamics of bacterial ribosome biogenesis

    No full text
    Supplement to hierarchical clustering of SSU r-protein and helix occupancy. Occupancy for each r-protein or helix calculated as described in Davis et al. 2016 (in revision). Data matrix clustered using average linkage and a Euclidian distance metric. Blocks 1-4 are labeled and colored. Structures labeled according to Table 1. RNA helices defined according to Supplemental Table 2

    Supplementary Figure 3 from Structure and dynamics of bacterial ribosome biogenesis

    No full text
    SSU folding blocks mapped to 16S secondary structure. 16S rRNA secondary structure map (Petrov et al., 2014) colored by domains and folding block as in Figure 3C. Contacts linking helix 36 to the central pseudoknot drawn with blue lines. Contacts linking block 2 drawn with orange lines. Remaining tertiary contacts drawn in black lines

    The Spectroscopic Basis of Fluorescence Triple Correlation Spectroscopy

    No full text
    We have developed fluorescence triple correlation spectroscopy (F3CS) as an extension of the widely used fluorescence microscopy technique fluorescence correlation spectroscopy. F3CS correlates three signals at once and provides additional capabilities for the study of systems with complex stoichiometry, kinetic processes, and irreversible reactions. A general theory of F3CS was developed to describe the interplay of molecular dynamics and microscope optics, leading to an analytical function to predict experimental triple correlations of molecules that freely diffuse through the tight focus of the microscope. Experimental correlations were calculated from raw fluorescence data using triple correlation integrals that extend multiple-tau correlation theory to delay times in two dimensions. The quality of experimental data was improved by tuning specific spectroscopic parameters and employing multiple independent detectors to minimize optoelectronic artifacts. Experiments with the reversible system of freely diffusing 16S rRNA revealed that triple correlation functions contain symmetries predicted from time-reversal arguments. Irreversible systems are shown to break these symmetries, and correlation strategies were developed to detect time-reversal asymmetries in a comprehensive way with respect to two delay times, each spanning many orders of magnitude in time. The correlation strategies, experimental approaches, and theory developed here enable studies of the composition and dynamics of complex systems using F3CS

    Composition of in vitro matured particles.

    No full text
    <p>Protein occupancy in 44/45S and 70S samples normalized to that of L20. Samples are colored in pairs with the 44/45S intermediate in a lighter shade than the 70S particle. Samples from strain 1043 (RbgA-F6A) are on the left (blue, green), those from strain 1055 (RbgA-F6A, L6-RC3) are on the right (orange, red). Semitransparent dots signify unique peptide measurements. The median value is denoted with a larger opaque marker. Protein L6 is highlighted in red. Proteins significantly depleted are colored orange.</p

    Proposed model for the role of RbgA in promoting late-stage large ribosome subunit assembly.

    No full text
    <p>A late assembly intermediate (LAI<sub>50-1</sub>) can proceed via two different pathways. Pathway 1 posits that RbgA binds prior to L6 (LAI<sub>50-2</sub>) while pathway 2 indicates L6 binds prior to RbgA (LAI<sub>50-3</sub>). When bound together (LAI<sub>50-4</sub>), RbgA facilitates proper interaction between L6 and the maturing ribosome, which triggers the incorporation of late ribosomal proteins. Once proper incorporation occurs, RbgA leaves the complex. The role of GTP hydrolysis in the assembly process is discussed in the text.</p

    <i>In vitro</i> maturation of large subunit intermediates.

    No full text
    <p><b>(A) <i>in vitro</i> maturation of 44S intermediate from RB1055.</b> Ribosome profile from cell lysate of strain RB1055 expressing mutated L6 protein (R3C) and RbgA-F6A protein after incubation at 0°C (blue) and 37°C (red) for 60 minutes. <b>(B) </b><b><i>in vitro</i></b><b> maturation of 45S intermediate from RB1043.</b> Ribosome profiles from cell lysate of strain RB1043 expressing RbgA-F6A protein and wild-type L6 protein after incubation at 0°C (blue) or 37°C (red) for 60 minutes. The X-axis indicates the direction of the profiles from the bottom of the gradient (43%) to the top of the gradient (18%). The Y-axis depicts absorbance at 260 nm, which is equivalent for both plots depicted. Dashed lines indicate the migration of the 70S, 50S, 44S and the 30S complexes in the gradient.</p

    Interaction between L6 protein and the 50S ribosomal subunit.

    No full text
    <p><b>A</b>. Crystal structure of 50S subunit from <i>E. coli</i> (PDB ID:2AW4) with the position of L6 indicated in blue. The location of late binding ribosomal proteins that are missing or highly reduced in the 45S particle are also highlighted (L16 (green), L28 (yellow) and L36 (cyan)} or highly reduced {L27 (orange), L33a (purple), L35 (red). <b>B</b>. L6 (blue) binding region including helix 97 (colored magenta) is shown in a magnified view and the residues mutated in suppressor strains are colored in red at the N terminal of L6 protein.</p

    Analysis of ribosome assembly in a L6 suppressor strains.

    No full text
    <p>The ribosome profiles of <i>rbgA</i>-F6A suppressor strains show an accumulation of a novel 44S complex. Ribosome profiles were analyzed from RB247 (wild-type cells), RB1043 (RbgA-F6A mutant), RB1051 (<i>rbgA</i>-F6A, <i>rplF</i>-R70P), RB1055 (<i>rbgA</i>-F6A, <i>rplF</i>-R3C), RB1057 (<i>rbgA</i>-F6A, <i>rplF</i>-H66L), RB1063 (<i>rbgA</i>-F6A, <i>rplF</i>-G5C), RB1065 (<i>rbgA</i>-F6A, <i>rplF</i>-G5S) and RB1068 (<i>rbgA</i>-F6A, <i>rplF</i>-T68R). Profiles were generated by sucrose density gradient centrifugation. Dashed lines indicate the migration of the 70S, 44S and the 30S subunits in the gradient.</p
    corecore