Complete list of canonical post-transcriptional modifications in the Bacillus subtilis ribosome and their link to RbgA driven large subunit assembly.

Ribosomal RNA modifications in prokaryotes have been sporadically studied, but there is a lack of a comprehensive picture of modification sites across bacterial phylogeny. Bacillus subtilis is a preeminent model organism for gram-positive bacteria, with a well-annotated and editable genome, convenient for fundamental studies and industrial use. Yet remarkably, there has been no complete characterization of its rRNA modification inventory. By expanding modern MS tools for the discovery of RNA modifications, we found a total of 25 modification sites in 16S and 23S rRNA of B. subtilis, including the chemical identity of the modified nucleosides and their precise sequence location. Furthermore, by perturbing large subunit biogenesis using depletion of an essential factor RbgA and measuring the completion of 23S modifications in the accumulated intermediate, we provide a first look at the order of modification steps during the late stages of assembly in B. subtilis. While our work expands the knowledge of bacterial rRNA modification patterns, adding B. subtilis to the list of fully annotated species after Escherichia coli and Thermus thermophilus, in a broader context, it provides the experimental framework for discovery and functional profiling of rRNA modifications to ultimately elucidate their role in ribosome biogenesis and translation.

Complete list of canonical post-transcriptional modifications in the Bacillus subtilis ribosome and their link to RbgA driven large subunit assembly.

Ribosomal RNA modifications in prokaryotes have been sporadically studied, but there is a lack of a comprehensive picture of modification sites across bacterial phylogeny. B. subtilis is a preeminent model organism for gram-positive bacteria, with a well-annotated and editable genome, convenient for fundamental studies and industrial use. Yet remarkably, there has been no complete characterization of its rRNA modification inventory. By expanding modern MS tools for the discovery of RNA modifications, we found a total of 25 modification sites in 16S and 23S rRNA of B. subtilis, including the chemical identity of the modified nucleosides and their precise sequence location. Furthermore, by perturbing large subunit biogenesis using depletion of an essential factor RbgA and measuring the completion of 23S modifications in the accumulated intermediate, we provide a first look at the order of modification steps during the late stages of assembly in B. subtilis. While our work expands the knowledge of bacterial rRNA modification patterns, adding B. subtilis to the list of fully annotated species after E. coli and T. thermophilus, in a broader context, it provides the experimental framework for discovery and functional profiling of rRNA modifications to ultimately elucidate their role in ribosome biogenesis and translation.

Three-State DNA Hairpin Conformational Dynamics Revealed by Higher-Order Fluorescence Correlation Spectroscopy.

Previous fluorescence correlation spectroscopy (FCS) measurements of DNA hairpin folding dynamics revealed at least three conformational states of the DNA are present, distinguished by the brightness of fluorescent dye-quencher labels. Rapid fluctuations between two of the states occurred on time scales observable by FCS. A third state that was static on the FCS time scale was also observed. In this study, we investigate these conformational states using newly developed higher-order FCS techniques. It is shown that conventional FCS alone cannot uniquely distinguish the conformational states or assign their roles in the observed mechanism. The additional information offered by higher-order FCS makes it possible (i) to uniquely identify the static and rapidly fluctuating states and (ii) to directly measure the brightnesses and populations of all three observed states. The rapid fluctuations occurring on the FCS time scale are due to a reversible reaction between the two lowest brightness levels, attributed to the folded and random-coil conformations of the DNA. Evidence is presented that the third state, which is the brightest, may be associated with spatially extended unfolded conformations that are isolated from the more compact conformations by a substantial barrier. These conformations attain a maximum equilibrium population of nearly 10% near physiological temperatures and salt concentrations.

Artifact-Free and Detection-Profile-Independent Higher-Order Fluorescence Correlation Spectroscopy for Microsecond-Resolved Kinetics. 1. Multidetector and Sub-Binning Approach.

Fluorescence correlation spectroscopy (FCS) is a powerful tool in the time-resolved analysis of nonreacting or reacting molecules in solution, based on fluorescence intensity fluctuations. However, conventional (second-order) FCS alone is insufficient to measure all parameters needed to describe a reaction or mixture, including concentrations, fluorescence brightnesses, and forward and reverse rate constants. For this purpose, correlations of higher powers of fluorescence intensity fluctuations can be calculated to yield additional information from the single-photon data stream collected in an FCS experiment. To describe systems of diffusing and reacting molecules, considering cumulants of fluorescence intensity results in simple expressions in which the reaction and diffusion parts factorize. The computation of higher-order correlations in experiments is hindered by shot-noise and common detector artifacts, the effects of which become worse with increasing order. In this article, we introduce a technique to calculate artifact-free higher-order correlation functions with improved time resolution, and without any need for modeling and calibration of detector artifacts. The technique is formulated for general multidetector experiments and verified in both two-detector and single-detector configurations. Good signal-to-noise ratio is achieved down to 1 µs in correlation curves up to order (2, 2). This capability makes possible a variety of new measurements including multicomponent analysis and fast reaction kinetics, as demonstrated in a companion article (10.1021/acs.jpcb.7b00408).

Artifact-Free and Detection-Profile-Independent Higher-Order Fluorescence Correlation Spectroscopy for Microsecond-Resolved Kinetics. 2. Mixtures and Reactions.

Fluorescence correlation spectroscopy (FCS) is a primary tool in the time-resolved analysis of nonreacting or reacting molecules in solution, based on fluorescence intensity fluctuations. However, conventional FCS alone is insufficient for a complete determination of reaction or mixture parameters. In an accompanying article, a technique for the computation of artifact-free higher-order correlations with microsecond time resolution was described. Here, we demonstrate the applications of the technique to analyze the systems of fast and slow reactions. As an example of non- or slow-reacting systems, the technique is applied to resolve two-component mixtures of labeled oligonucleotides. Next, the protonation reaction of fluorescein isothiocyanate in phosphate buffer is analyzed as an example of fast reactions <10 µs (actual time scale ∼6 µs). By reference to an (apparent) nonreacting system, the simple factorized form of cumulant-based higher-order correlations is exploited to remove the dependence on the molecular detection function (MDF). Therefore, there is no need to model and characterize the experimental MDF, and the precision and the accuracy of the technique are enhanced. It is verified that the higher-order correlation analysis enables a complete and simultaneous determination of the number and brightness parameters of mixing or reacting molecules, the reaction relaxation time, and forward and reverse reaction rates.