Analysis of mutations at residues A2451 and G2447 of 23S rRNA in the peptidyltransferase active site of the 50S ribosomal subunit
Analysis of mutations at residues A2451 and G2447 of 23S rRNA in the peptidyltransferase active site of the 50S ribosomal subunit
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DOI：
10.1073/pnas.151257098
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发表时间：
2001-07-31
影响因子：
11.1
通讯作者：
Dahlberg, AE
中科院分区：
文献类型：
--
作者：
Thompson, J;Kim, DF;Dahlberg, AE
基于最近的原子分辨率的X射线结构的50 S核糖体亚基，残基A2451和G2447的235 rRNA被提出直接参与核糖体催化的肽带的形成。我们已经研究了在这些核苷酸携带突变的核糖体的肽基转移酶和蛋白质合成活性。在大肠杆菌收费，纯突变体核糖体人口携带G2447 A或G2447 C突变保持细胞活力。在体外，G2447 A核糖体支持蛋白质合成的速率与野生型核糖体相当。在单营业额肽基转移酶测定，G2447 A核糖体被证明具有基本上未受损的肽基转移酶活性在饱和底物浓度。在普遍保守的A2451的所有三个碱基的变化赋予了一个显性致死表型时，在E，toll表达。尽管如此，大量的2451突变体核糖体积累在多聚核糖体中，并且所有三个2451突变刺激了体内终止密码子的移码和通读。此外，携带A2451 U颠换的核糖体在体外合成全长β-内酰胺酶链。通过从体外转录的235 rRNA重建嗜热脂肪芽孢杆菌50 S亚基，产生在A2451处具有变化的纯突变体核糖体群体。在单周转肽基转移酶测定中，这些突变使肽键形成的速率降低了3至14倍。肽基转移酶活性和体外β-内酰胺酶合成的核糖体与突变的A2451或G2447是高度耐氯霉素。具有A2451和G2447突变的核糖体的肽基转移酶活性的显著水平需要与这些残基在催化中的作用相协调。
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.