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Functional genetic selection of Helix 66 in Escherichia coli 23S rRNA identified the eukaryotic-binding sequence for ribosomal protein L2.

Kitahara K, Kajiura A, Sato NS, Suzuki T - Nucleic Acids Res. (2007)

Bottom Line: The isolated variants consisted of eukaryotic sequences, in addition to prokaryotic sequences.These results suggest that L2/L8e does not recognize a specific base sequence of H66, but rather a characteristic architecture of H66.The growth phenotype of the isolated variants correlated well with their ability of subunit association.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

ABSTRACT
Ribosomal protein L2 is a highly conserved primary 23S rRNA-binding protein. L2 specifically recognizes the internal bulge sequence in Helix 66 (H66) of 23S rRNA and is localized to the intersubunit space through formation of bridge B7b with 16S rRNA. The L2-binding site in H66 is highly conserved in prokaryotic ribosomes, whereas the corresponding site in eukaryotic ribosomes has evolved into distinct classes of sequences. We performed a systematic genetic selection of randomized rRNA sequences in Escherichia coli, and isolated 20 functional variants of the L2-binding site. The isolated variants consisted of eukaryotic sequences, in addition to prokaryotic sequences. These results suggest that L2/L8e does not recognize a specific base sequence of H66, but rather a characteristic architecture of H66. The growth phenotype of the isolated variants correlated well with their ability of subunit association. Upon continuous cultivation of a deleterious variant, we isolated two spontaneous mutations within domain IV of 23S rRNA that compensated for its weak subunit association, and alleviated its growth defect, implying that functional interactions between intersubunit bridges compensate ribosomal function.

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Sequences of the genetically selected functional variants of the N6-region in H66. (A) Secondary structures of the consensus class I L2-binding site, and the 11 class I variants. Consensus base-triple nucleotides (C1800, G1817 and A1819) in each variant are boxed. The inset shows the chemical structure of the base-triple. (B) Secondary structures of the consensus class II L2-binding site, and the 9 class II variants. Consensus base-triple nucleotides (A1800, U1817 and A1819) in each variant are boxed. The inset shows the chemical structure of the base-triple.
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Figure 2: Sequences of the genetically selected functional variants of the N6-region in H66. (A) Secondary structures of the consensus class I L2-binding site, and the 11 class I variants. Consensus base-triple nucleotides (C1800, G1817 and A1819) in each variant are boxed. The inset shows the chemical structure of the base-triple. (B) Secondary structures of the consensus class II L2-binding site, and the 9 class II variants. Consensus base-triple nucleotides (A1800, U1817 and A1819) in each variant are boxed. The inset shows the chemical structure of the base-triple.

Mentions: According to phylogenetic analysis of rRNAs from all living organisms, there are two distinct classes of L2-binding sites in H66 (see Figure 2A and B) (28)(www.rna.icmb.utexas.edu). In the crystal structures of bacterial ribosomes, a characteristic base-triple composed of C1800, G1817 and A1819 is visible in the L2-binding site of H66 (see Figure 1C) (1,5). In addition, a G1799·U1818 pair also stacks to this base-triple, and participates in a hydrogen (H)-bond network to form a core structure in this region. This characteristic core structure of the L2-binding site appears to induce a kinked conformation of H66 (see Figure 4A). This type of base-triple, designated as class I binding site, is conserved in bacteria, archea, mitochondria and chloroplasts. In eukaryotic rRNAs, the corresponding bases are replaced by A1800, U1817 and A1819 (E. coli numbering). Assuming that eukaryotic L2/L8e-binding sites also have a similar structure as the class I binding site, these three bases are likely to form a characteristic base-triple (Figure 2B), which is designated as class II binding site (see Figure 2B). The class II binding site is highly conserved in eukaryotic rRNAs, while all bacteria-type ribosomes have the class I binding site. Since L8e can replace bacterial L2 in E. coli cell, these distinct classes of sequences appear to provide functionally similar binding sites for L2/L8e proteins.Figure 1.


Functional genetic selection of Helix 66 in Escherichia coli 23S rRNA identified the eukaryotic-binding sequence for ribosomal protein L2.

Kitahara K, Kajiura A, Sato NS, Suzuki T - Nucleic Acids Res. (2007)

Sequences of the genetically selected functional variants of the N6-region in H66. (A) Secondary structures of the consensus class I L2-binding site, and the 11 class I variants. Consensus base-triple nucleotides (C1800, G1817 and A1819) in each variant are boxed. The inset shows the chemical structure of the base-triple. (B) Secondary structures of the consensus class II L2-binding site, and the 9 class II variants. Consensus base-triple nucleotides (A1800, U1817 and A1819) in each variant are boxed. The inset shows the chemical structure of the base-triple.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1919495&req=5

Figure 2: Sequences of the genetically selected functional variants of the N6-region in H66. (A) Secondary structures of the consensus class I L2-binding site, and the 11 class I variants. Consensus base-triple nucleotides (C1800, G1817 and A1819) in each variant are boxed. The inset shows the chemical structure of the base-triple. (B) Secondary structures of the consensus class II L2-binding site, and the 9 class II variants. Consensus base-triple nucleotides (A1800, U1817 and A1819) in each variant are boxed. The inset shows the chemical structure of the base-triple.
Mentions: According to phylogenetic analysis of rRNAs from all living organisms, there are two distinct classes of L2-binding sites in H66 (see Figure 2A and B) (28)(www.rna.icmb.utexas.edu). In the crystal structures of bacterial ribosomes, a characteristic base-triple composed of C1800, G1817 and A1819 is visible in the L2-binding site of H66 (see Figure 1C) (1,5). In addition, a G1799·U1818 pair also stacks to this base-triple, and participates in a hydrogen (H)-bond network to form a core structure in this region. This characteristic core structure of the L2-binding site appears to induce a kinked conformation of H66 (see Figure 4A). This type of base-triple, designated as class I binding site, is conserved in bacteria, archea, mitochondria and chloroplasts. In eukaryotic rRNAs, the corresponding bases are replaced by A1800, U1817 and A1819 (E. coli numbering). Assuming that eukaryotic L2/L8e-binding sites also have a similar structure as the class I binding site, these three bases are likely to form a characteristic base-triple (Figure 2B), which is designated as class II binding site (see Figure 2B). The class II binding site is highly conserved in eukaryotic rRNAs, while all bacteria-type ribosomes have the class I binding site. Since L8e can replace bacterial L2 in E. coli cell, these distinct classes of sequences appear to provide functionally similar binding sites for L2/L8e proteins.Figure 1.

Bottom Line: The isolated variants consisted of eukaryotic sequences, in addition to prokaryotic sequences.These results suggest that L2/L8e does not recognize a specific base sequence of H66, but rather a characteristic architecture of H66.The growth phenotype of the isolated variants correlated well with their ability of subunit association.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

ABSTRACT
Ribosomal protein L2 is a highly conserved primary 23S rRNA-binding protein. L2 specifically recognizes the internal bulge sequence in Helix 66 (H66) of 23S rRNA and is localized to the intersubunit space through formation of bridge B7b with 16S rRNA. The L2-binding site in H66 is highly conserved in prokaryotic ribosomes, whereas the corresponding site in eukaryotic ribosomes has evolved into distinct classes of sequences. We performed a systematic genetic selection of randomized rRNA sequences in Escherichia coli, and isolated 20 functional variants of the L2-binding site. The isolated variants consisted of eukaryotic sequences, in addition to prokaryotic sequences. These results suggest that L2/L8e does not recognize a specific base sequence of H66, but rather a characteristic architecture of H66. The growth phenotype of the isolated variants correlated well with their ability of subunit association. Upon continuous cultivation of a deleterious variant, we isolated two spontaneous mutations within domain IV of 23S rRNA that compensated for its weak subunit association, and alleviated its growth defect, implying that functional interactions between intersubunit bridges compensate ribosomal function.

Show MeSH
Related in: MedlinePlus