Fig 1.
RING-MaP single-molecule correlated chemical probing and network analysis of a large RNA.
(A) Overview of the RING-MaP experiment and network analysis. DMS chemical adducts on an RNA are detected by MaP, which records adduct sites as mutations and indels in the cDNA sequence generated by reverse transcription. Mutations co-occurring on the same read are analyzed for correlations that report RINGs, which are then visualized using a network graph. (B) Workflow for network analysis and detection of structural communities. Network analysis was performed using Gephi. DMS, dimethyl sulfate; MaP, mutational profiling; RING, RNA interaction group; RING-MaP, RNA interaction groups analyzed by mutational profiling.
Fig 2.
Spc binding site in the 16S rRNA in E. coli cells revealed by DMS footprinting.
(A) Per-nucleotide DMS-induced mutation rate profiles for 16S rRNA in cells treated with Rif (+Rif) and with Rif and Spc (+Spc). The plot shows a 550-nucleotide region spanning the 3' domain of the 16S rRNA; a single nucleotide at C1192 had a notable difference between the two experiments. The underlying data for this figure are available at https://doi.org/10.6084/m9.figshare.9252995.v1. (B) Model of Spc bound near C1192 (in red) of the 16S rRNA. (C) Structure of the small ribosomal subunit. 16S rRNA is light purple, ribosomal proteins are brown, mRNA is green, tRNA is black, and Spc is yellow. 30S subunits domains are labeled by morphology. Ribosome structure in all figures are from PDB 4v56 and 5afi [5,12]. DMS, dimethyl sulfate; PDB, Protein Data Bank; Rif, rifampicin; rRNA, ribosomal RNA; Spc, spectinomycin.
Fig 3.
Network analysis applied to RING-MaP correlation data reveals that Spc induces extensive changes in through-space structural communication in the ribosome.
(A) Correlation of RING-MaP data for independent experiments based on Spc-untreated versus Spc-treated samples (top panel; both samples pretreated with Rif); and between biological replicates (bottom panel) (B–C) Network analysis of structural communication in the 16S rRNA in (B) the absence and (C) the presence of Spc. Network analysis reveals four structural communities (blue, red, yellow, and green). Nodes are colored by community and are sized based on the number of correlations with other nodes in the network. Edges indicate internucleotide correlations, and edge weights indicate correlation strength. For both–Spc and +Spc conditions, cells were pretreated with Rif, which allows the rRNAs to fully assemble into complete subunits [22]. (D) Superposition of network nodes on the three-dimensional structure of the 30S subunit. Nodes were categorized by strength into strong, medium, and weak indicated by large, medium, and small spheres, respectively; strong interactions are shown as colored lines. (E) Edges that were strengthened (left) or weakened (right) upon addition of Spc. The underlying data for this figure are available at https://doi.org/10.6084/m9.figshare.9252995.v1. Rif, rifampicin; RING-MaP, RNA interaction groups analyzed by mutational profiling; rRNA, ribosomal RNA; Spc, spectinomycin.
Fig 4.
Domain architecture of the 16S rRNA as defined by network communities.
Spheres indicate strong network nodes, colored by community. (A) 16S rRNA domain architecture based on secondary structure. Note that part of the central (platform) domain, as defined by the 16S rRNA secondary structure, is a structural component of the body. (B) Network communities correctly define the body domain and the more compact platform domain (yellow). These domains emerged naturally from the network analysis, even though no three-dimensional structural information was included in the network analysis. (C) The conventional head domain contains two RING network communities occupying distinct regions, defined here as the outer-head domain (red) and the inner-head and spine domain (blue). (D) Small-subunit domain model based on RING network communities in the 16S rRNA. The domains shown correspond to RING-MaP network community nodes of high correlation observed for the +Spc (+Rif) treatment condition; however, the same communities are observed in the absence of Spc. Rif, rifampicin; RING, RNA interaction group; RING-MaP, RNA interaction groups analyzed by mutational profiling; rRNA, ribosomal RNA; Spc, spectinomycin.
Fig 5.
Cross-community interactions strengthened upon Spc binding.
(A, B) Cross-community correlations connecting the outer-head (red) and inner-head and spine (blue) communities are shown as grey lines on secondary structure diagrams for experiments performed (A) without and (B) with Spc treatment. The strongest cross-community interactions link nodes in the outer-head domain at the h34-h35-h38 3-helix junction to nodes in helix 44 of the inner-head and spine domain. Orange spheres indicate the two hinges that allow head domain swiveling [10]. (C) Cross-community interactions visualized with respect to the three-dimensional structure of bacterial ribosome. The two dashed lines mark the axes for head swiveling (passing through hinges 1 and 2) and for intersubunit rotation (at bridge B3). Bridge B3 links helix 44 of 30S rRNA with helix 71 of the 23S rRNA of the large subunit. rRNA, ribosomal RNA; Spc, spectinomycin.
Fig 6.
Effect of Spc on ribosome dynamics.
Spc binding blocks free swiveling of the head domain and halts intersubunit rotation due to strengthened through-space interactions between pivot regions, thereby trapping the ribosome in a partially swiveled state. Black dashed lines indicate movements involving head swiveling and L1 stalk movement during translocation. Red dashed lines outline the portion of the outer-head domain concealed behind the inner-head domain. Spc, spectinomycin.