Visualization of conformational variability in the domains of long single-stranded RNA molecules

Figures

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  Figure 1.. AFM imaging of 28S rRNA. (A) A gallery of 300 × 300 nm2 images of individual 28S rRNA molecules. (B) Selected images demonstrating the length of skeleton representations of each molecule in comparison to the longest end-to-end main chain. Lines are dilated to widths of three pixels for ease of visualization. (C) Histograms demonstrating the distribution of the lengths of the skeleton representations (left) and the main chains (middle) for N = 23 molecules. The degree of branching (right) was measured by taking the skeleton length divided by the main chain length.

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  Figure 2.. Domain identification in 28S rRNA. (A) A method of generating profiles along the main chain of the molecule (left) can be used to quantify the height of pixels immediately underlying the main chain or can be used to quantify the local volume along the main chain by summing pixels in the region of the molecule which are in the vicinity of each main chain pixel (middle). The correspondence of peaks in the volume profile to structures along the molecule are denoted by colored arrows (middle and right). (B) Analogous structures in images of 23 molecules are denoted by colored arrowheads as in (A). Here each molecule is named using a descriptor, ‘mol’, followed by the length of the main chain in nanometers. (C) The cumulative volume in the profiles was used to estimate the number of nucleotides in each domain by using a conversion factor assuming that the total volume of each molecule corresponds to the total number of nucleotides (5070 nt) in the molecule. The prominent domains identified in (A) and (B) were assigned names according to their general morphology: including sY (small Y)-red, bc (big claw)-orange, bY (big Y)-green, boot-blue and sc (small claw)-purple. Regions…

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  Figure 3.. Comparison of AFM-based 28S rRNA structures to previously reported 3D-based structures. (A) Representative AFM images of the 28S rRNA with domains identified in Figure 2 (arrows). (B) Image of the 3D-based secondary structure of Homo sapiens 28S rRNA (11,12) generated with ribovision (10) showing how the predicted nucleotide ranges from our analysis (Table 1) coincide with predicted secondary structures of the 28S rRNA molecules from our AFM images shown on the right. Domains are color-coded as the arrows in (A). Helices and eukaryotic-specific expansion segments (ES) of interest are labeled, in addition to a kink-turn motif reported to exist within helix 42 (22,23) and the location of the peptidyl transferase center (PTC). For a more detailed description of the helices and nucleotide numbering, please refer to the aforementioned references (11,12). (C) Diagram of the conserved domains identified in AFM images and the predicted number of nucleotides comprising each domain. The five prominent domains identified in (A) are color-coded as in (A), in addition to 4 smaller domains shown in black.

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  Figure 4.. Representative zoomed images (100 × 100 nm2) of each prominent domain identified in Figure 2 in addition to each interdomain region. The regions are as follows: (A) 5′end, (B) sY, (C) sY→bc, (D) bc, (E) bc→bY, (F) bY, (G) bY→boot, (H) boot, (I) boot→sc and (J) sc. The three branches of the bc (D) are labeled with colored arrowheads from the one which appears most 5′ to the one which appears most 3′. In each set of interdoman images (A,C,E,G,I), the prominent domains are labeled with asterisks in order to delineate the interdomain boundaries. (A) Representative structures of the 5′ end with the sY structure labeled with a red asterisk. (C) Representative images of the sY→bc region with the sY and bc structures labeled with red and yellow asterisks, respectively. The two main structures observed in this region are labeled with colored arrows. (E) Representative images of the bc→bY region with the bc and bY structures labeled with yellow and green asterisks, respectively. The two main structures observed in this region are labeled with colored arrows. (G) Representative images of the bY→boot region with the bY and boot structures labeled with green and blue asterisks, respectively.…

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  Figure 5.. Representative zoomed images of the interdomain associations observed in some subsets of the molecules. (A) 170 × 170 nm2 images demonstrating a complex structure in the region where the bc structure is generally observed (see Figure 2B). The sY and bY structures bordering this structure are labeled with red and green asterisks, respectively. (B) 146 × 146 nm2 images demonstrating an association between the boot (blue arrow) and the bY (green arow) structure (in contrast to the bY→boot region observed in Figure 4G). An additional small structure extending opposite from the boot is also labeled with a yellow arrow. (C) 146 × 146 nm2 images demonstrating an association between the boot and the sc structure (in contrast to the boot→sc region observed in Figure 4I). The upstream bY structure is labeled with a green asterisk.