Porse B T, Garrett R A
Institute of Molecular Biology, University of Copenhagen, Denmark.
J Mol Biol. 1995 May 26;249(1):1-10. doi: 10.1006/jmbi.1995.0276.
Random mutations were generated in the lower half of the peptidyl transferase loop in domain V of 23 S rRNA from Escherichia 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 transferase 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-G2582, 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 m6A2503G 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.
使用聚合酶链反应（PCR）方法、一种用于鉴定突变体的快速程序和基于质粒的表达系统，在大肠杆菌23S rRNA结构域V的肽基转移酶环下半部分产生随机突变。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掺入核糖体以及突变体核糖体的肽基转移酶活性的影响。当含有染色体编码的23S rRNA的核糖体被红霉素抑制时，14个突变体表现出病态或非常病态，这一发现强调了整个区域对肽基转移酶中心的普遍重要性，并且除其中一个外，所有这些突变体在其突变的核糖体中都表现出低水平的肽基转移酶活性。两个产生无活性核糖体的突变，即psi 2580→C和U2584→G，被指定到供体底物结合位点，并提出了肽基-tRNA的3'-末端序列与psi/U-G-G2582序列之间可能的碱基配对相互作用，该序列在所有非线粒体23S样rRNA序列中都是保守的。三个与氨酰-tRNA结合有关的位点发生了突变：突变体m6A2503G产生无活性的核糖体，而突变体Um2552A/C和U2555C的核糖体分别产生低活性和正常活性。三个突变体U2528C、G2550A和A2565U为肽基转移酶中心发生的构象重排提供了证据，这种重排可能受亚基-亚基相互作用的影响。其他产生在肽基转移酶活性方面严重缺陷的核糖体的突变体是U2493A、U2493C、A2497G、A2530G、G2557A和A2589G。
