[논문]Functional Analysis of the Residues C770 and G771 of E. coli 16S rRNA Implicated in Forming the Intersubunit Bridge B2c of the Ribosome

Kim, Hong-Man; Yeom, Ji-Hyun; Ha, Hye-Jung; Kim, Jong-Myung; Lee, Kang-Seok

Functional Analysis of the Residues C770 and G771 of E. coli 16S rRNA Implicated in Forming the Intersubunit Bridge B2c of the Ribosome 원문보기

Journal of microbiology and biotechnology, v.17 no.7, 2007년, pp.1204 - 1207

Kim, Hong-Man (Department of Life Science, Chung-Ang University) , Yeom, Ji-Hyun (Department of Life Science, Chung-Ang University) , Ha, Hye-Jung (Department of Life Science, Chung-Ang University) , Kim, Jong-Myung (Department of Life Science, Chung-Ang University) , Lee, Kang-Seok (Department of Life Science, Chung-Ang University)

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Structural analyses have shown that nucleotides at the positions 770 and 771 of Escherichia coli 16S rRNA are implicated in forming one of highly conserved intersubunit bridges of the ribosome, B2c. To examine a functional role of these residues, base substitutions were introduced at these positions and mutant ribosomes were analyzed for their protein synthesis ability using a specialized ribosome system. The results showed requirement of a pyrimidine at the position 770 for ribosome function regardless of the nucleotide identity at the position 771. Sucrose gradient profiles of ribosomes revealed that the loss of protein-synthesis ability of mutant ribosome bearing a base substitution from C to G at the position 770 stems from its inability to form 70S ribosomes. These findings indicate involvement of nucleotide at the position 770, not 771, in ribosomal subunit association and provide a useful rRNA mutation that can be used as a target to investigate the physical interaction between 16S and 23S rRNA.

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The extension reaction contained a mixture of dATP, dCTP, dTTP, and ddGTP. The synthesized cDNAs were resolved by PAGE and the ratios of mutant to utant rRNA were determined by comparing the amount of radioactivity in each of the two bands.
ability. To test this hypothesis, mutant ribosomes bearing a base substitution at the position 770 were purified using a sucrose gradient and analyzed for their ability to form 70S ribosome. C₇₇0U and C₇₇0G mutations were chosen for this experiment because ribosomes bearing C₇₇0U and C₇₇0G showed the highest and lowest protein synthesis function, respectively among mutant ribosomes with a point mutation at this position.

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Functional analysis of mutations constructed at the positions 770 and 771 of 16S rRNA in pRNA122. A. Nucleotide identities are indicated in the order of 770 and 771, and mutations are underlined. B.
To test this hypothesis, mutant ribosomes bearing a base substitution at the position 770 were purified using a sucrose gradient and analyzed for their ability to form 70S ribosome. C₇₇0U and C₇₇0G mutations were chosen for this experiment because ribosomes bearing C₇₇0U and C₇₇0G showed the highest and lowest protein synthesis function, respectively among mutant ribosomes with a point mutation at this position. Sucrose gradient profiles of mutant ribosomes prepared from cells expressing 16S rRNA bearing C₇₇0G (16S- C₇₇0G) showed an increased abundance of 30S subunits compared to those from cells expressing 16S-C₇₇0U.
The resuIts showed that a base substitution from C (wild-type) to U at the position 770 (C₇₇0U) resulted in mildly deleterious effect (MIC=500) whereas a change to a purine resulted in moderately (C₇₇0A, MIC=400) or strongly deleterious effect (C₇₇0G, MIC=150) on ribosome function, indicating that a pyrimidine (C or U) at the position 770 is required for ribosome function (Fig. 1). A base substitution at the position 771 had no effect (MIC of G771 A=600) or mildly deleterious effect (MIC of G771C and G771Ue500 and 550, respectively) on the protein synthesis ability of the ribosome.

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Functional Analysis of the Residues C770 and G771 of E. coli 16S rRNA Implicated in Forming the Intersubunit Bridge B2c of the Ribosome 원문보기

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Functional Analysis of the Residues C770 and G771 of E. coli 16S rRNA Implicated in Forming the Intersubunit Bridge B2c of the Ribosome 원문보기

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