Porse B T, Thi-Ngoc H P, Garrett R A
RNA Regulation Centre, Institute of Molecular Biology, University of Copenhagen, Denmark.
J Mol Biol. 1996 Dec 6;264(3):472-83. doi: 10.1006/jmbi.1996.0655.
An RNA region associated with the donor substrate site, located at the base of the peptidyl transferase loop of 23 S rRNA, was subjected to a comprehensive single-site mutational study. Growth phenotypes of Escherichia coli cells were characterized on induction of synthesis of the mutated rRNAs and the mutated ribosomes were tested, selectively, for their capacity to generate peptide bonds under the conditions of the "fragment" assay. Most of the mutants exhibited dominant or recessive lethal growth phenotypes and, in general, defective growth correlated with low activities in peptide bond formation, although exceptions were observed with normal growth and low activities, and vice versa. All these phenotypes are consistent with defects occurring in the structure of the ribosomal donor site and/or the capacity of the donor substrate to enter or leave this site. A compensating base change approach was employed to test for Watson-Crick base-pairing interactions between the -CCA end of the P-site bound tRNA(Phe) and this region of the peptidyl-transferase loop. Single nucleotide substitutions were introduced into the -CCA end of tRNA(Phe) and the ability of the 3'-terminal pentanucleotide fragments to act as donor substrates was examined for ribosomes carrying the different mutated 23 S rRNAs. No evidence was found for the occurrence of Watson-Crick base-pairing interactions. However, the data are consistent with the formation of a Hoogsteen pair between the 3'-terminal adenosine base of the donor substrate and U2585 of the 23 S rRNA.
一个与供体底物位点相关的RNA区域，位于23 S rRNA肽基转移酶环的基部，接受了全面的单位点突变研究。对大肠杆菌细胞的生长表型进行了表征，即在诱导合成突变的rRNA时进行观察，并对突变的核糖体进行测试，在“片段”测定条件下选择性地检测它们形成肽键的能力。大多数突变体表现出显性或隐性致死生长表型，一般来说，生长缺陷与肽键形成活性低相关，不过也观察到了正常生长但活性低以及活性低但生长正常的例外情况。所有这些表型都与核糖体供体位点结构中的缺陷和/或供体底物进入或离开该位点的能力缺陷一致。采用补偿碱基变化方法来测试P位点结合的tRNA（Phe）的-CCA末端与肽基转移酶环的该区域之间的沃森-克里克碱基配对相互作用。将单核苷酸取代引入tRNA（Phe）的-CCA末端，并检查3'-末端五核苷酸片段作为供体底物对携带不同突变23 S rRNA的核糖体的作用能力。未发现沃森-克里克碱基配对相互作用发生的证据。然而，数据与供体底物的3'-末端腺苷碱基与23 S rRNA的U2585之间形成Hoogsteen配对一致。
