Mutations at nucleotides G2251 and U2585 of 23 S rRNA perturb the peptidyl transferase center of the ribosome
Mutations at nucleotides G2251 and U2585 of 23 S rRNA perturb the peptidyl transferase center of the ribosome
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DOI：
10.1006/jmbi.1996.0780
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发表时间：
1997-02-14
影响因子：
5.6
通讯作者：
Noller, HF
中科院分区：
文献类型：
--
作者：
Green, R;Samaha, RR;Noller, HF
先前的实验表明，23 S rRNA的遗传学保守的G2252与肽基tRNA的C74形成Watson-Crick碱基对。在这里介绍的研究中，定点突变被引入到23 S rRNA中的另外两个保守位置，G2251和U2585，这两个位置先前与tRNA的CCA受体末端与50 S亚基P位点的相互作用有关。通过测定（1）体内表型，（2）突变体核糖体体外结合tRNA寡核苷酸片段的能力（使用等位基因特异性引物延伸足迹法）和（3）突变体核糖体催化肽键形成的能力（使用嵌合重建方法）表征突变体23 S rRNA。当突变型23 S rRNA与内源性野生型23 S rRNA共表达时，任一位置的突变均赋予显性致死表型。2585处的突变破坏野生型（CCA）tRNA寡核苷酸片段的结合，并导致重组核糖体的肽基转移酶活性适度降低。相比之下，2251处的突变消除了野生型（CCA）tRNA片段的结合和使用野生型tRNA片段的肽基转移酶活性。在这两种情况下，结合或肽基转移酶活性的损失抑制突变的tRNA寡核苷酸片段。化学修饰分析表明，2251位的突变扰乱了2584至2586位碱基的反应性，进一步证明了23 S rRNA的2250环与23 S rRNA结构域V中央环的2585区直接或间接相互作用。(C)出版社：Academic Press Limited。
Previous experiments have shown that the phylogenetically conserved G2252 of 23 S rRNA forms a Watson-Crick base-pair with C74 of peptidyl-tRNA. In the studies presented here, site-directed mutations were introduced at two other conserved positions in 23 S rRNA, G2251 and U2585, that were previously implicated in interaction of the CCA acceptor end of tRNA with the 50 S subunit P site. The mutant 23 S rRNAs were characterized by determining (1) the in vivo phenotypes, (2) the ability of mutant ribosomes to bind tRNA oligonucleotide fragments in vitro, using footprinting with allele-specific primer extension and (3) the ability of mutant ribosomes to catalyze peptide bond formation using a chimeric reconstitution approach. Mutations at either position confer a dominant lethal phenotype when the mutant 23 S rRNA is coexpressed with the endogenous wild-type 23 S rRNA. Mutations at 2585 disrupt binding of the wild-type (CCA) tRNA oligonucleotide fragment and cause a modest decrease in the peptidyl transferase activity of reconstituted ribosomes. By contrast, mutations at 2251 abolish both binding of the wild-type (CCA) tRNA fragment and peptidyl transferase activity using the wild-type tRNA fragment. In neither case was the loss of binding or peptidyl transferase activity suppressed by mutations in the tRNA oligonucleotide fragment. Chemical modification analysis revealed that mutations at 2251 perturb the reactivity of bases 2584 to 2586, providing further evidence that the 2250 loop of 23 S rRNA interacts, either directly or indirectly, with the 2585 region in the central loop of domain V of 23 S rRNA. (C) 1997 Academic Press Limited.