Is the in situ accessibility of the 16S rRNA of Escherichia coli for Cy3-labeled oligonucleotide probes predicted by a three-dimensional structure model of the 30S ribosomal subunit?

Abstract

Systematic studies on the hybridization of fluorescently labeled, rRNA-targeted oligonucleotides have shown strong variations in in situ accessibility. Reliable predictions of target site accessibility would contribute to more-rational design of probes for the identification of individual microbial cells in their natural environments. During the past 3 years, numerous studies of the higher-order structure of the ribosome have advanced our understanding of its spatial conformation. These studies range from the identification of rRNA-rRNA interactions based on covariation analyses to physical imaging of the ribosome for the identification of protein-rRNA interactions. Here we reevaluate our Escherichia coli 16S rRNA in situ accessibility data with regard to a tertiary-structure model of the small subunit of the ribosome. We localized target sequences of 176 oligonucleotides on a 3.0-A-resolution three-dimensional (3D) model of the 30S ribosomal subunit. Little correlation was found between probe hybridization efficiency and the proximity of the probe target region to the surface of the 30S ribosomal subunit model. We attribute this to the fact that fluorescence in situ hybridization is performed on fixed cells containing denatured ribosomes, whereas 3D models of the ribosome are based on its native conformation. The effects of different fixation and hybridization protocols on the fluorescence signals conferred by a set of 10 representative probes were tested. The presence or absence of the strongly denaturing detergent sodium dodecyl sulfate had a much more pronounced effect than a change of fixative from paraformaldehyde to ethanol.

FIG. 1.

Target sequences of fluorescently labeled oligonucleotide probes are shown within a 3D structure model of the 30S ribosomal subunit of E. coli. Ribosomal proteins are shown in blue. Red (A), orange (B), yellow (C), green (D), light blue (E), and magenta (F) indicate target sequences belonging to probe brightness classes I (highest fluorescence signal) to VI (lowest fluorescence signal), respectively, defined in the study of Behrens et al. (4).

FIG. 2.

3D structure model of the 30S ribosomal subunit of E. coli. (A) The target region of class I probe Eco907 (positions 907 to 925) is shown in red; it comprises the 5′ end of helix 30, helix 2, and the 3′ end of helix 31 (helix numbering according to the work of Brosius et al. [7]). (B) Highlighted in red is the target region of class VI probe Eco621 (positions 621 to 638), corresponding to the loop region of helix 22. Proteins are shown in blue.

FIG. 3.

Predicted secondary structures of E. coli 16S rRNA (9). Numbers attached to nucleotides indicate sequence numbering; boldfaced numbers indicate helix numbering according to the work of Brosius et al. (7). (A) Regions of the 16S rRNA of E. coli contacted by the ribosomal proteins S2 to S20 are highlighted. Proteins S1 and S21 are not included. Actual contacts as observed in the crystal structure of the 30S ribosomal subunit of T. thermophilus are shown with colored circles around the RNA residues in question. No distinction was made between backbone-only contacts, base-only contacts, and contacts with both the backbone and the base. (B) Distribution of relative fluorescence hybridization intensities of 176 oligonucleotide probes targeting the 16S rRNA of E. coli. The different colors indicate different brightness classes, as explained in the key.

FIG. 4.

Detailed view of the transition zone between the 5′ end of helix 23 and helix 24 (helix numbering according to the work of Brosius et al. [7]) within the 30S ribosomal subunit of E. coli. (A) Overview of the whole 30S subunit, with the region to be shown in large scale highlighted in red. Proteins are depicted as blue tubes. (B) Target region of probe Eco668 (positions 668 to 685) marked as a ball-and-stick model. Blue tubes, ribosomal proteins. (C) Same as panel B but without ribosomal proteins.

FIG. 5.

Comparison of fluorescence intensities of Cy3-labeled oligonucleotide probes hybridized with or without SDS in the hybridization buffer to differently fixed E. coli cells. Fluorescence intensity is expressed as a percentage of that obtained with standard beads. Dark shaded bars, ethanol fixation, storage in an ethanol-1× PBS mixture, and standard hybridization with 0.01% SDS. Solid bars, PFA fixation, storage in an ethanol-1× PBS mixture, and standard hybridization with 0.01% SDS. Open bars, PFA fixation, storage in an ethanol-1× PBS mixture, and hybridization without SDS. Light shaded bars, PFA fixation, storage in 1× PBS, and standard hybridization with 0.01% SDS.