Comparative 3-D modeling of tmRNA

Abstract

Background: Trans-translation releases stalled ribosomes from truncated mRNAs and tags defective proteins for proteolytic degradation using transfer-messenger RNA (tmRNA). This small stable RNA represents a hybrid of tRNA- and mRNA-like domains connected by a variable number of pseudoknots. Comparative sequence analysis of tmRNAs found in bacteria, plastids, and mitochondria provides considerable insights into their secondary structures. Progress toward understanding the molecular mechanism of template switching, which constitutes an essential step in trans-translation, is hampered by our limited knowledge about the three-dimensional folding of tmRNA.

Results: To facilitate experimental testing of the molecular intricacies of trans-translation, which often require appropriately modified tmRNA derivatives, we developed a procedure for building three-dimensional models of tmRNA. Using comparative sequence analysis, phylogenetically-supported 2-D structures were obtained to serve as input for the program ERNA-3D. Motifs containing loops and turns were extracted from the known structures of other RNAs and used to improve the tmRNA models. Biologically feasible 3-D models for the entire tmRNA molecule could be obtained. The models were characterized by a functionally significant close proximity between the tRNA-like domain and the resume codon. Potential conformational changes which might lead to a more open structure of tmRNA upon binding to the ribosome are discussed. The method, described in detail for the tmRNAs of Escherichia coli, Bacillus anthracis, and Caulobacter crescentus, is applicable to every tmRNA.

Conclusion: Improved molecular models of biological significance were obtained. These models will guide in the design of experiments and provide a better understanding of trans-translation. The comparative procedure described here for tmRNA is easily adopted for the modeling the members of other RNA families.

Figure 1

Secondary structure of E. coli tmRNA. Phylogenetically-supported helices are highlighted in gray and numbered from 1 to 12. The 5' and 3' ends are indicated. Arrows represent connections from 5' to 3'. Residues are numbered in increments of ten. Weakly supported regions and basepairs are shown in boxes. The disproved potential pairing of C109 with G136 is labeled with an open arrowhead. The star labels the first nucleotide of the resume codon. The tag peptide sequence is shown below the mRNA-like region. The stop codons are indicated with solid arrowheads. Three domains are distinguished: the tRNA-like domain (TLD), the mRNA-like domain (MLD), and the pseudoknot domain (PKD).

Figure 2

Motif modeling procedure. Motifs, for example the nonamer-loop shown in the top-left panel, were identified in the known high-resolution structures (top-right) with the help of SCOR [12]. The PDB coordinates were extracted (bottom-right) and compared with the 3-D model generated by ERNA-3D (bottom-left) to deduce relevant models.

Figure 3

3-D model of Escherichia coli tmRNA. The 3-D model of Escherichia coli tmRNA is viewed as a ribbon diagram from the side in panel A, the top in panel B, and in panel C turned by approximately 90° around the y-axis in relation to A. Panel D shows a representation of the corresponding 2-D structure using the identical coloring scheme. Labeled are the 5' and 3' ends, the resume (R) and stop codons (S), and the three domains (TLD, MLD, PKD). The figure was produced with iMol [53] and the PDB coordinates of additional file 7: Ecoli-closed.pdb.

Figure 4

3-D model of Bacillus anthracis tmRNA. The 3-D model of Bacillus anthracis tmRNA is viewed as a ribbon diagram from the side in panel A, the top in panel B, and in panel C turned by approximately 90° around the y-axis in relation to A. Panel D shows a representation of the corresponding 2-D structure using the identical coloring scheme. Labeled are the 5' and 3' ends, the resume (R) and stop codons (S), and the three domains (TLD, MLD, PKD). The figure was produced with iMol [53] and the PDB coordinates of additional file 9: Banthracis-closed.pdb.

Figure 5

3-D model of Caulobacter crescentus tmRNA. The 3-D model of Caulobacter crescentus s tmRNA is viewed as a ribbon diagram from the side in panel A, the top in panel B, and in panel C turned by approximately 90° around the y-axis in relation to A. Panel D shows a representation of the corresponding 2-D structure using the identical coloring scheme. Labeled are the 5' and 3' ends, the resume (R) and stop codons (S), and the three domains (TLD, MLD, PKD). The figure was produced with iMol [53] and the PDB coordinates of additional file 10: Ccrescentus-closed.pdb.

Figure 6

Conformational changes in Escherichia coli tmRNA. Panel A: closed form of the E. coli model as shown in Figure 3. Panel B: open conformation adjusted to more closely resemble the ribosome-bound form as determined by cryo-EM [10] using additional file 8: Ecoli-open.pdb. Panel C: coordinates extracted from the cryo-EM model [10]. The TLD is shown in dark purple, helix 2 in green, pk1 in yellow, helix 5 in pink, pk2 in turquoise, pk3 in red, and pk4 in dark blue. Panel D: Electron density map of the 50S subunit in light blue, the 30S subunit in yellow, and the bound tmRNA (in the absence of ribosomal protein S1) in dark blue (from [10]).