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Structural insights into the role of rRNA modifications in protein synthesis and ribosome assembly.

Polikanov YS, Melnikov SV, Söll D, Steitz TA - Nat. Struct. Mol. Biol. (2015)

Bottom Line: We report crystal structures of the Thermus thermophilus ribosome at 2.3- to 2.5-Å resolution, which have enabled modeling of rRNA modifications.The structures reveal contacts of modified nucleotides with mRNA and tRNAs or protein pY, and contacts within the ribosome interior stabilizing the functional fold of rRNA.Our work provides a resource to explore the roles of rRNA modifications and yields a more comprehensive atomic model of a bacterial ribosome.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA. [2] Howard Hughes Medical Institute at Yale University, New Haven, Connecticut, USA.

ABSTRACT
We report crystal structures of the Thermus thermophilus ribosome at 2.3- to 2.5-Å resolution, which have enabled modeling of rRNA modifications. The structures reveal contacts of modified nucleotides with mRNA and tRNAs or protein pY, and contacts within the ribosome interior stabilizing the functional fold of rRNA. Our work provides a resource to explore the roles of rRNA modifications and yields a more comprehensive atomic model of a bacterial ribosome.

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Related in: MedlinePlus

Electron density maps allow comprehensive modeling of rRNA and ribosomal protein modifications of the 30S subunit(a) Spatial distribution of modified nucleotides in the structure of the small ribosomal subunit from T. thermophilus. Here, in Fig. 2 and throughout the text rRNA nucleotides are named according to the E. coli numbering system. Panels (b-i) show unbiased difference Fo-Fc electron density maps for eight types of modifications present in 16S rRNA and ribosomal proteins S12 and S4. Grey mesh shows the Fo-Fc map after refinement with the entire modified nucleotides or amino acid residues omitted (contoured at 2.7-3.0σ). Green mesh shows the Fo-Fc electron density map after refinement with unmodified nucleotides (contoured at 1.5-2.5σ). The modifications shown are: (b) N3-methyluridine 1498, (c) N4,O2’-dimethylcytidine 1402, (d) N2-methylguanosine 1207, (e) N2-dimethylguanosine 966, (f) N7-methylguanosine 527, (g) N6-dimethyladenosine 1519; (h) β-methylthioaspartate of protein S12, (i) 4Fe-4S iron-sulfur cluster of protein S4.
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Figure 1: Electron density maps allow comprehensive modeling of rRNA and ribosomal protein modifications of the 30S subunit(a) Spatial distribution of modified nucleotides in the structure of the small ribosomal subunit from T. thermophilus. Here, in Fig. 2 and throughout the text rRNA nucleotides are named according to the E. coli numbering system. Panels (b-i) show unbiased difference Fo-Fc electron density maps for eight types of modifications present in 16S rRNA and ribosomal proteins S12 and S4. Grey mesh shows the Fo-Fc map after refinement with the entire modified nucleotides or amino acid residues omitted (contoured at 2.7-3.0σ). Green mesh shows the Fo-Fc electron density map after refinement with unmodified nucleotides (contoured at 1.5-2.5σ). The modifications shown are: (b) N3-methyluridine 1498, (c) N4,O2’-dimethylcytidine 1402, (d) N2-methylguanosine 1207, (e) N2-dimethylguanosine 966, (f) N7-methylguanosine 527, (g) N6-dimethyladenosine 1519; (h) β-methylthioaspartate of protein S12, (i) 4Fe-4S iron-sulfur cluster of protein S4.

Mentions: Here, we set out to apply our recent advances in crystal treatment to reach diffraction resolution suitable for visualization of modified nucleotides within the 70S ribosome (see Online Methods). As a result, we report two crystal structures of the T. thermophilus (Tth) 70S ribosome reflecting two functional states – hibernating ribosomes, in complex with the hibernation factor pY, and translating ribosomes, in complex with mRNA and tRNAs, – determined at 2.3Å and 2.5Å resolution, respectively (I/σI=1) (Supplementary Table 1). The resolution at which I/σI=2 is 2.5Å and 2.7Å, respectively. At this resolution methylation of ribose and nucleotide bases can be directly visualized in the unbiased electron density maps (Fig. 1, panels d-j and m-q). Pseudouridines cannot be distinguished from uridines and, therefore, their modeling was guided by biochemical data4. In one particular case, however, the electron density map confirmed the presence of pseudouridine (Ψ2605), consistent with hydrogen bonding between the base and the phosphate via a water molecule, which is impossible for uridine base (Fig. 2b). Apart from rRNA modifications, the maps also confirmed the presence of a modification in ribosomal protein S12 – β-methyl-thiolation of residue Asp88 (Fig. 1h, Supplementary Fig. 1a, b), – and a prosthetic group in ribosomal protein S4 – the 4Fe-4S iron-sulfur cluster, coordinated by cysteine residues (Fig. 1i, Supplementary Fig. 1c, d). In total, each of our models contains 23 modified RNA nucleotides – all known modifications of Tth rRNA, – among which 5 are conserved across bacteria, archaea and eukaryotes, 18 are common between Tth and E. coli, and 5 are specific to Tth (Fig. 1a, Fig. 2a, Supplementary Table 1).


Structural insights into the role of rRNA modifications in protein synthesis and ribosome assembly.

Polikanov YS, Melnikov SV, Söll D, Steitz TA - Nat. Struct. Mol. Biol. (2015)

Electron density maps allow comprehensive modeling of rRNA and ribosomal protein modifications of the 30S subunit(a) Spatial distribution of modified nucleotides in the structure of the small ribosomal subunit from T. thermophilus. Here, in Fig. 2 and throughout the text rRNA nucleotides are named according to the E. coli numbering system. Panels (b-i) show unbiased difference Fo-Fc electron density maps for eight types of modifications present in 16S rRNA and ribosomal proteins S12 and S4. Grey mesh shows the Fo-Fc map after refinement with the entire modified nucleotides or amino acid residues omitted (contoured at 2.7-3.0σ). Green mesh shows the Fo-Fc electron density map after refinement with unmodified nucleotides (contoured at 1.5-2.5σ). The modifications shown are: (b) N3-methyluridine 1498, (c) N4,O2’-dimethylcytidine 1402, (d) N2-methylguanosine 1207, (e) N2-dimethylguanosine 966, (f) N7-methylguanosine 527, (g) N6-dimethyladenosine 1519; (h) β-methylthioaspartate of protein S12, (i) 4Fe-4S iron-sulfur cluster of protein S4.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4401423&req=5

Figure 1: Electron density maps allow comprehensive modeling of rRNA and ribosomal protein modifications of the 30S subunit(a) Spatial distribution of modified nucleotides in the structure of the small ribosomal subunit from T. thermophilus. Here, in Fig. 2 and throughout the text rRNA nucleotides are named according to the E. coli numbering system. Panels (b-i) show unbiased difference Fo-Fc electron density maps for eight types of modifications present in 16S rRNA and ribosomal proteins S12 and S4. Grey mesh shows the Fo-Fc map after refinement with the entire modified nucleotides or amino acid residues omitted (contoured at 2.7-3.0σ). Green mesh shows the Fo-Fc electron density map after refinement with unmodified nucleotides (contoured at 1.5-2.5σ). The modifications shown are: (b) N3-methyluridine 1498, (c) N4,O2’-dimethylcytidine 1402, (d) N2-methylguanosine 1207, (e) N2-dimethylguanosine 966, (f) N7-methylguanosine 527, (g) N6-dimethyladenosine 1519; (h) β-methylthioaspartate of protein S12, (i) 4Fe-4S iron-sulfur cluster of protein S4.
Mentions: Here, we set out to apply our recent advances in crystal treatment to reach diffraction resolution suitable for visualization of modified nucleotides within the 70S ribosome (see Online Methods). As a result, we report two crystal structures of the T. thermophilus (Tth) 70S ribosome reflecting two functional states – hibernating ribosomes, in complex with the hibernation factor pY, and translating ribosomes, in complex with mRNA and tRNAs, – determined at 2.3Å and 2.5Å resolution, respectively (I/σI=1) (Supplementary Table 1). The resolution at which I/σI=2 is 2.5Å and 2.7Å, respectively. At this resolution methylation of ribose and nucleotide bases can be directly visualized in the unbiased electron density maps (Fig. 1, panels d-j and m-q). Pseudouridines cannot be distinguished from uridines and, therefore, their modeling was guided by biochemical data4. In one particular case, however, the electron density map confirmed the presence of pseudouridine (Ψ2605), consistent with hydrogen bonding between the base and the phosphate via a water molecule, which is impossible for uridine base (Fig. 2b). Apart from rRNA modifications, the maps also confirmed the presence of a modification in ribosomal protein S12 – β-methyl-thiolation of residue Asp88 (Fig. 1h, Supplementary Fig. 1a, b), – and a prosthetic group in ribosomal protein S4 – the 4Fe-4S iron-sulfur cluster, coordinated by cysteine residues (Fig. 1i, Supplementary Fig. 1c, d). In total, each of our models contains 23 modified RNA nucleotides – all known modifications of Tth rRNA, – among which 5 are conserved across bacteria, archaea and eukaryotes, 18 are common between Tth and E. coli, and 5 are specific to Tth (Fig. 1a, Fig. 2a, Supplementary Table 1).

Bottom Line: We report crystal structures of the Thermus thermophilus ribosome at 2.3- to 2.5-Å resolution, which have enabled modeling of rRNA modifications.The structures reveal contacts of modified nucleotides with mRNA and tRNAs or protein pY, and contacts within the ribosome interior stabilizing the functional fold of rRNA.Our work provides a resource to explore the roles of rRNA modifications and yields a more comprehensive atomic model of a bacterial ribosome.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA. [2] Howard Hughes Medical Institute at Yale University, New Haven, Connecticut, USA.

ABSTRACT
We report crystal structures of the Thermus thermophilus ribosome at 2.3- to 2.5-Å resolution, which have enabled modeling of rRNA modifications. The structures reveal contacts of modified nucleotides with mRNA and tRNAs or protein pY, and contacts within the ribosome interior stabilizing the functional fold of rRNA. Our work provides a resource to explore the roles of rRNA modifications and yields a more comprehensive atomic model of a bacterial ribosome.

Show MeSH
Related in: MedlinePlus