Analysis of mutations at residues A2451 and G2447 of 23S rRNA in the peptidyltransferase active site of the 50S ribosomal subunit

被引:165
作者
Thompson, J [1 ]
Kim, DF
O'Connor, M
Lieberman, KR
Bayfield, MA
Gregory, ST
Green, R
Noller, HF
Dahlberg, AE
机构
[1] Brown Univ, Dept Mol & Cell Biol & Biochem, Providence, RI 02912 USA
[2] Johns Hopkins Univ, Sch Med, Dept Mol Biol & Genet, Howard Hughes Med Inst, Baltimore, MD 21205 USA
[3] Univ Calif Santa Cruz, Sinsheimer Labs, Ctr Mol Biol RNA, Santa Cruz, CA 95064 USA
关键词
D O I
10.1073/pnas.151257098
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
On the basis of the recent atomic-resolution x-ray structure of the 50S ribosomal subunit, residues A2451 and G2447 of 235 rRNA were proposed to participate directly in ribosome-catalyzed peptide band formation. We have examined the peptidyltransferase and protein synthesis activities of ribosomes carrying mutations at these nucleotides. In Escherichia toll, pure mutant ribosome populations carrying either the G2447A or G2447C mutations maintained cell viability. In vitro, the G2447A ribosomes supported protein synthesis at a rate comparable to that of wild-type ribosomes. In single-turnover peptidyltransferase assays, G2447A ribosomes were shown to have essentially unimpaired peptidyltransferase activity at saturating substrate concentrations. All three base changes at the universally conserved A2451 conferred a dominant lethal phenotype when expressed in E, toll. Nonetheless, significant amounts of 2451 mutant ribosomes accumulated in polysomes, and all three 2451 mutations stimulated frameshifting and readthrough of stop codons in vivo. Furthermore, ribosomes carrying the A2451U transversion synthesized full-length beta -lactamase chains in vitro. Pure mutant ribosome populations with changes at A2451 were generated by reconstituting Bacillus stearothermophilus 50S subunits from in vitro transcribed 235 rRNA. In single-turnover peptidyltransferase assays, the rate of peptide bond formation was diminished 3- to 14-fold by these mutations. Peptidyltransferase activity and in vitro beta -lactamase synthesis by ribosomes with mutations at A2451 or G2447 were highly resistant to chloramphenicol. The significant levels of peptidyltransferase activity of ribosomes with mutations at A2451 and G2447 need to be reconciled with the roles proposed for these residues in catalysis.
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页码:9002 / 9007
页数:6
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