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首页> 外文期刊>Journal of Molecular Biology >MAPPING IMPORTANT NUCLEOTIDES IN THE PEPTIDYL TRANSFERASE CENTRE OF 23 S RRNA USING A RANDOM MUTAGENESIS APPROACH
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MAPPING IMPORTANT NUCLEOTIDES IN THE PEPTIDYL TRANSFERASE CENTRE OF 23 S RRNA USING A RANDOM MUTAGENESIS APPROACH

机译：使用随机诱变方法在23 S RRNA的肽基转移酶中心映射重要的核苷酸

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Random mutations were generated in the lower half of the peptidyl transferase loop in domain V of 23 S rRNA from Escheuichin coli using a polymerase chain reaction (PCR) approach, a rapid procedure for identifying mutants and a plasmid-based expression system. The effects of 21 single-site mutations, at 18 different positions, on cell growth, mutant rRNA incorporation into ribosomes and peptidyl tranferase activity of the mutant ribosomes were analysed. The general importance of the whole region for the peptidyl transferase centre was emphasized by the finding that 14 of the mutants were sick, or very sick, when ribosomes containing chromosomal-encoded 23 S rRNA were inhibited by erythromycin, and all except one of these exhibited low levels of peptidyl transferase activity in their mutated ribosomes. Two mutations, Psi 2580 --> C and U2584 --> G that both yielded inactive ribosomes were assigned to the donor substrate binding site and a possible base-pairing interaction between the 3'-terminal sequence of the peptidyl-tRNA and the sequence Psi/U-G-G(2582), that is conserved in all the non-mitochondrial 23 S-like rRNA sequences, is proposed. Three sites that have been implicated in aminoacyl-tRNA binding were mutated: mutant m(6)A2503G yielded inactive ribosomes, while ribosomes from mutants Um2552A/C and U2555C yielded low and normal activities, respectively. Three mutants, U2528C, G2550A and A2565U, provide evidence for conformational rearrangements occurring in the peptidyl transferase centre which may be affected by the subunit-subunit interaction. Other mutants which yielded ribosomes that were seriously defective in peptidyl transferase activity were U2493A, U2493C, A2497G, A2530G, G2557A and A2589G. [References: 43]
机译：使用聚合酶链反应（PCR）方法，鉴定突变体的快速步骤和基于质粒的表达系统，在来自大肠杆菌的23 S rRNA的结构域V的肽基转移酶环的下半部分中产生随机突变。分析了18个不同位置的21个单点突变对细胞生长，突变体rRNA掺入核糖体的影响以及突变核糖体的肽基转移酶活性。这一发现强调了整个区域对于肽基转移酶中心的普遍重要性，因为当含有染色体编码的23 S rRNA的核糖体被红霉素抑制时，其中的14个突变体有病或非常有病，并且除其中之一外，所有这些都表现出突变核糖体中低水平的肽基转移酶活性。两种均产生无活性核糖体的Psi 2580-> C和U2584-> G突变被指定给供体底物结合位点，以及肽基tRNA 3'-末端序列与该序列之间可能的碱基配对相互作用提出了在所有非线粒体23 S样rRNA序列中都保守的Psi / UGG（2582）。已突变涉及氨酰-tRNA结合的三个位点：突变体m（6）A2503G产生非活性核糖体，而来自突变体Um2552A / C和U2555C的核糖体分别产生低活性和正常活性。 U2528C，G2550A和A2565U这三个突变体为可能在亚基-亚基相互作用中影响的肽基转移酶中心构象重排提供了证据。产生在肽基转移酶活性上严重缺陷的核糖体的其他突变体是U2493A，U2493C，A2497G，A2530G，G2557A和A2589G。 [参考：43]

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