Lieberman K R, Dahlberg A E
Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912.
J Biol Chem. 1994 Jun 10;269(23):16163-9.
We have constructed the double mutant G2252C/G2253C in Escherichia coli 23 S rRNA by site-directed mutagenesis. These phylogenetically conserved residues are protected from chemical modification by the 3' CCA terminus of the peptidyl-tRNA site (P site)-bound tRNA. Expression of C2252/C2253 23 S rRNA in E. coli severely compromises cell growth. Mutant rRNA is assembled into 50 S subunits and 70 S ribosomes but is discriminated against in polysomes. Mutant ribosomes function at lower rates in peptidyltransferase assays than wild type ribosomes. To test whether this defect derives from disruption of base pairing with the 2 cytidines of the invariant 3' CCA terminus of tRNA, a mutant E. coli tRNAPhe gene was constructed, with the CCA sequence changed to GGA. As deacylated species, mutant and wild type tRNAPhe inhibit peptidyl transfer identically. Mutant tRNAPhe was aminoacylated in vitro but failed to react as a P site substrate, with either mutant or wild type ribosomes. These results support a role for G2252 and G2253 of 23 S rRNA in peptidyltransferase function and a role for the 3' residues of peptidyl-tRNA in catalytically productive P site interaction; but they fail to provide evidence supporting canonical base pairing between these 23 S residues and the 3' end of peptidyl-tRNA.
我们通过定点诱变在大肠杆菌23 S rRNA中构建了双突变体G2252C/G2253C。这些在系统发育上保守的残基受到肽基-tRNA位点（P位点）结合的tRNA的3' CCA末端的化学修饰保护。C2252/C2253 23 S rRNA在大肠杆菌中的表达严重损害细胞生长。突变rRNA组装成50 S亚基和70 S核糖体，但在多核糖体中受到歧视。在肽基转移酶测定中，突变核糖体的功能速率低于野生型核糖体。为了测试这种缺陷是否源于与tRNA不变的3' CCA末端的2个胞嘧啶碱基配对的破坏，构建了一个突变的大肠杆菌tRNAPhe基因，将CCA序列改为GGA。作为去酰化物种，突变型和野生型tRNAPhe对肽基转移的抑制作用相同。突变型tRNAPhe在体外被氨酰化，但无论是与突变型还是野生型核糖体反应，都不能作为P位点底物。这些结果支持23 S rRNA的G2252和G2253在肽基转移酶功能中的作用，以及肽基-tRNA的3' 残基在催化性有效P位点相互作用中的作用；但它们未能提供证据支持这些23 S残基与肽基-tRNA的3' 末端之间的规范碱基配对。
