Cunningham P R, Richard R B, Weitzmann C J, Nurse K, Ofengand J
Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110.
Biochimie. 1991 Jun;73(6):789-96. doi: 10.1016/0300-9084(91)90058-9.
16S RNA of Escherichia coli lacking all post-transcriptional modifications and with 5'-termini of pppGGGAGA-, pppGAA-, pppAAA-, and pAAA- were prepared by in vitro transcription of appropriately engineered plasmids with T7 or SP6 RNA polymerases. These synthetic versions of 16S RNA were compared with natural 16S RNA for their ability to reconstitute 30S ribosomal subunits in vitro using varied conditions for both the isolation of the RNA and for reconstitution. Under all conditions studied, natural 16S RNA assembled correctly, as judged by velocity centrifugation comparison with an internal standard of native 30S particles, and the recovered ribosomes were 80-100% as active as native 30S ribosomes in initiation complex formation, P site binding of AcVal-tRNA, A site binding of Phe-tRNA, and formation of the first peptide bond. In contrast, all of the synthetic constructs including pAAA-, which has the same sequence as native 16S RNA, were only partially active in reconstitution and in the functional assays. We conclude that the lack of the 10 methylated nucleotides and/or the 2 pseudouridylate residues present in natural 16S RNA must be responsible for the reduced activity of the synthetic RNAs in ribosome assembly and function.
通过用T7或SP6 RNA聚合酶对经过适当改造的质粒进行体外转录，制备了缺乏所有转录后修饰且5'末端分别为pppGGGAGA -、pppGAA -、pppAAA -和pAAA -的大肠杆菌16S RNA。将这些合成的16S RNA与天然16S RNA进行比较，考察它们在不同的RNA分离条件和重构条件下体外重构30S核糖体亚基的能力。在所有研究条件下，通过与天然30S颗粒内标进行速度离心比较判断，天然16S RNA能正确组装，回收的核糖体在起始复合物形成、AcVal - tRNA的P位点结合、Phe - tRNA的A位点结合以及第一个肽键形成方面的活性是天然30S核糖体的80 - 100%。相比之下，所有合成构建体，包括与天然16S RNA序列相同的pAAA -，在重构和功能测定中仅部分具有活性。我们得出结论，天然16S RNA中缺少的10个甲基化核苷酸和/或2个假尿苷残基必定是合成RNA在核糖体组装和功能方面活性降低的原因。
