tmRNA

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tmRNA (also known as 10Sa RNA) stands for transfer-messenger-RNA. The gene encoding the tmRNA is ssrA. It is found in all bacterial genomes that have been sequenced, and is an important part of translation regulation. To remain stable, tmRNA associates with Small Protein B (SmpB).

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Modeling the tmRNA structure in the ribosome

For modeling of the tmRNA-2 structure in the ribosome we used the published ribosome X-ray structure with a defined mRNA path (Yusupova et al, 2006). We have assumed that the mRNA part of tmRNA should occupy the same mRNA binding region on the ribosome as the normal mRNA. The stop codon in this complex should be at the A-site and the preceding codon involved in a codon-anticodon interaction with tRNA should occupy the P-site. The TLD of tmRNA should be at the ribosomal E-site region and should keep the structure of the acceptor arm of tRNA. The position of the L1 stalk was taken from (Harms et al, 2001). Since the arch consisting of tmRNA pseudoknots and helices was observed by cryo-EM in the pre-initiation complex (Valle et al, 2003) as well as in the tmRNA-4 complex described here, we suggest that such a structure should be present at all steps of the tmRNA path through the ribosome. The individual pseudoknot structures were either taken from the cryo-EM model (Gillet et al, 2007) or derived from NMR data for a similar pseudoknot (Nonin-Lecomte, Felden, and Dardel 2006). According to our probing data all stable secondary structure elements should exist in the resulting model. As a simplification of the system each nucleotide or amino acid is represented by one atom. Only the above mentioned secondary structure elements of tmRNA were fixed and the other parts were unstructured at the beginning of the analysis. The program allowed the formation of the tmRNA structure with known secondary structure elements in the empty space in the ribosome or at the ribosome surface. The structure was optimized by energy minimization and manual corrections.

The resulting structural model for tmRNA-2 inside the ribosome is shown in Fig. 8A (pdb to download).

The model is in good agreement with the chemical probing data. The SmpB protein occupies the site on TLD as known from its X-ray structure in complex with tmRNA fragment (Gutmann et al, 2003). The TLD is located at the ribosomal E-site. The protected A79-A86 loop is located at the entrance of the mRNA binding pocket; pK1 is nearby at the E-site tRNA exit channel. Helix ?? that determines the location of the L1 stalk is moved from its position in the original X-ray structure (Yusupova et al, 2006) as suggested by cryo-EM data for conformational changes in the ribosome caused by exiting deacylated tRNA []. Only in this case is there enough space between the subunits at the L1 side of the ribosome in order to accommodate the TLD with SmpB and pK1 simultaneously. Helices 2a, 2b and 2c support the TLD location at the E-site, helix 2d creates a link between pK1 and the arch consisting of pK4, pK2 and pK3. The arch is surrounding the head of the 30S subunit starting from the shoulder; helix 5 is located at the entrance to the mRNA channel and can be easily unwound during subsequent steps of ORF translation without influencing the arch structure. The resume codon is at the P-site of the ribosome and the 2^nd ORF codon is at the A site. One can see that all protected bases in the single-stranded regions are indeed involved in the interactions with the ribosome and the position of the bases with enhanced reactivity corresponds to possible distortions in the RNA chain.

The same approach was applied to create a model for tmRNA-4 in the ribosome (Fig. 8B, pdb to download).

Again, there is a good agreement between the chemical probing data, cryo-electron tomography data and the proposed model. At this step of trans-translation the TLD-SmpB complex is moved out from the ribosome and is located at the platform side of the 30S subunit. It is not tightly fixed on the ribosome and can occupy any position such that it does not interfere with the deacylated tRNA that leaves the ribosome through the exit site. Helices 2a, 2b and 2c link TLD to pK4 (part of the arch) on one side and pK1 and the A79-A86 loop on the other side supported by helix 2d. This helix could be partially unwound, but hidden in the ribosome. It is thus protected from chemical modification even more than in tmRNA being in solution. The protected A79-A86 loop together with the first nucleotide in the resume codon is tightly bound in the mRNA binding pocket (Yusupova et al, 2006). Its position is fixed on the ribosome by pK1. The arch that can be visualized by cryo-electron tomography consists of three pseudoknots. This arch is surrounding the head of the 30S subunit starting from the shoulder, as in the case of tmRNA-2. It is clearly seen that the size of the arch in the model is reduced in comparison with the pre-initiation state (Fig. 8C) that corresponds to our tomography data (Fig. 8D). The model allows movement of the arch around the head of the 30S subunit. Helix 5 is located at the entrance to the mRNA channel. The 3^rd and 4^th codons of the tmRNA ORF are located at the P- and A-site positions, as determined by X-ray analysis (REF), in the decoding center on the 30S subunit. Again, all protected bases in the single-stranded regions are indeed involved in the interactions with the ribosome and the position of the bases with enhanced reactivity corresponds to possible distortions in the RNA chain.

Since the modeling approach described above allows the generation of models that are in agreement with experimental data, we decided to apply it for modeling the process of template switching during trans-translation. To model the initiation complex with the acceptor arm of TLD at the A-site and tRNA bound to the previous mRNA at the A-site, we used the X-ray structure of the ribosome with tRNAs bound to A- and P-site. This is under the assumption that TLD should have the conformation of the acceptor arm of tRNA at the A-site and the mRNA part of tmRNA should be in the mRNA channel. After application of the structure formation approach described above we obtained the model presented in Fig. 9A. One can see that there is no room for pK1 in the decoding area of the ribosome and pK1 is located at the intersubunit space on the L7/L12 side. In the pre-initiation complex the SmpB protein was shown to move out its binding site on TLD in solution. In the initiation complex the only space where it is possible to place SmpB is pK1 and the loop formed by a single-stranded RNA region (A79-G87) is….

The model for the next step of trans-translation is presented in Fig. 9B. At this stage EFG dependent translocation has taken place and the TLD of tmRNA has moved to the P-site and the resume codon appears at the A-site. One can see that at this stage the only possibility for SmpB, pK1 and the A79-G87 loop to be in the ribosome is to occupy the space in the E-site area on the 30S subunit.

At the next step TLD enters the ribosomal E-site and SmpB can easily move to its binding site on TLD as shown for tmRNA-2 in the ribosomal complex in Fig. 8A.

-- Main.AndreyGolovin - 01 Dec 2008