High-resolution structure of the Escherichia coli ribosome

Protein synthesis by the ribosome is highly dependent on ions. Moreover, the ribosome harbors posttranscriptional and posttranslational modifications, the function of which is poorly understood. Here, we present a high-resolution structure of the E. coli ribosome that suggests how solvation contributes to ribosome integrity and helps to explain the phylogenetic conservation of key elements including post-transcriptional and post-translational modifications.

HFSP Long-Term Fellow Jonas Noeske and colleagues
authored on Tue, 24 March 2015

Protein synthesis by the ribosome is highly dependent on the ionic conditions in the cellular environment. Due to the polyanionic character of the ribosome, it harbors a large number of cations, which influence key functional steps like mRNA decoding, translocation, and intersubunit rotation. Furthermore, the ribosomal RNA (rRNA) and ribosomal proteins undergo numerous posttranscriptional and posttranslational modifications respectively. In addition to the conversion of uridine to pseudouridine these posttranscriptional modifications include methylation of rRNA nucleobases and of the 2'-hydroxy group of the ribose. Such modifications lead to altered electrostatic potential of the rRNA nucleotides, which results in altered base-pairing potentials, preferred sugar puckers, and different base-stacking properties. Interestingly, most of these ribosomal modifications are clustered in key functional sites of the ribosome, for example the peptidyl transferase center (PTC) and the decoding center.

Figure: A high-resolution structure reveals solvation properties as well as posttranscriptional and posttranslational modifications of the intact E. coli 70S ribosome.

Our high-resolution structure of the intact 70S ribosome of E. coli reveals solvation at key functional sites such as antibiotic binding sites and ribosomal bridges, the contact points between the small 30S and the large 50S ribosomal subunit. We identified several water molecules that buttress ribosomal bridge B3, which is considered the 'pivot point' of intersubunit rotation. Furthermore, a number of solvent molecules overlap with the binding site of clinically important antibiotics like macrolides, ketolides and streptogramin B. These solvent molecules could be included in future computational docking methods aiming at identifying new, more potent antibiotics.

A number of ribosomal modifications cluster near the PTC. We can identify that methylation of the 2'-hydroxy group of G2251 of the P loop of the large subunit rRNA stabilizes its sugar pucker in the C3'-endo conformation, which may be important to properly position the acceptor arm of the P-site transfer RNA (tRNA) for the peptidyl transferase reaction. All other ribosomal nucleotides carrying a 2' methyl group are also stabilized in the C3'-endo pucker in the new ribosome structure. Protein uS12 of the small subunit forms parts of the mRNA-decoding center. Its proline at position 45 is identified to assume a cis-peptide conformation, which has functional implications for mRNA decoding mRNA and tRNA translocation. Due to its position immediately next to the mRNA, hydroxylation of the corresponding proline in its eukaryotic ribosomal protein homologue affects stop-codon readthrough in a sequence- and context-dependent manner. The geometry of this proline in the bacterial ribosome suggests a model for how its hydroxylation could influence the energetics of mRNA decoding and mRNA and tRNA translocation.

For most of the pseudouridines we observe a water-mediated contact between the nucleobase and its phosphate backbone, which is consistent with the model that pseudouridines stabilize local ribosomal structure. We also identified 15 pyrimidines in the energetically unfavorable syn conformation of which many are supported by phylogenentic analysis as being nearly universal in bacteria or across all domains of life. This reveals conserved base-pair patterns, which could contribute to rRNA tertiary folding and key ribosomal functions, such as tRNA translocation.

Finally, the new high-resolution structure of the E. coli 70S ribosome can be used as a resource and reference for comparison to other RNA structures and future ribosome structures in different conformational states, for analyses of ribosomal phylogenetics and for biochemical studies.

Reference

High-resolution structure of the Eschericia coli ribosome. Jonas Noeske, Michael R Wasserman, Daniel S Terry, Roger B Altman, Scott C Blanchard, Jamie H D Cate. Nature Structural and Molecular Biology, doi 10.1038/nsmb.2994.

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