Samaha R R, O'Brien B, O'Brien T W, Noller H F
Sinsheimer Laboratories, University of California, Santa Cruz 95064.
Proc Natl Acad Sci U S A. 1994 Aug 16;91(17):7884-8. doi: 10.1073/pnas.91.17.7884.
Small (30S) subunits of Escherichia coli ribosomes are composed of 21 proteins and a 1542-nucleotide 16S rRNA, whose secondary structure is divided into three domains. An in vitro transcript of the 3' domain of 16S rRNA (residues 923-1542), assembles efficiently with 30S ribosomal proteins to form a compact ribonucleoprotein (RNP) particle. Isolated particles examined under the electron microscope have a globular appearance, similar in size and shape to the head of the 30S ribosomal subunit. Two-dimensional gel analysis of the particles indicates the presence of proteins S3, S7, S9, S10, S13, S14, and S19 and smaller amounts of S2, all of which have been localized to the head of the 30S subunit by immunoelectron microscopy and neutron diffraction and belong to the S7 assembly family. Interestingly, protein S4, which is believed to interact exclusively with the 5' domain, is also reproducibly found associated with the particles in significant amounts. Chemical probing of the RNA in the assembled particle reveals characteristic cleavage protection patterns, showing that the proteins assemble with the 3'-domain RNA similarly to the way in which they assemble with 16S rRNA, although some of the later steps of assembly appear to be incomplete. These results show that the 3' domain of 16S rRNA can indeed assemble independently of the rest of the 30S subunit into a particle that resembles its structure in the ribosome. In addition, the assembled particles are able to bind spectinomycin with an affinity comparable to that of 30S subunits.
大肠杆菌核糖体的小（30S）亚基由21种蛋白质和一个1542个核苷酸的16S rRNA组成，其二级结构分为三个结构域。16S rRNA 3'结构域（923 - 1542位残基）的体外转录本能与30S核糖体蛋白高效组装，形成紧密的核糖核蛋白（RNP）颗粒。在电子显微镜下观察分离出的颗粒呈球状，大小和形状与30S核糖体亚基的头部相似。颗粒的二维凝胶分析表明存在蛋白质S3、S7、S9、S10、S13、S14和S19以及少量的S2，所有这些蛋白质通过免疫电子显微镜和中子衍射已定位到30S亚基的头部，且属于S7组装家族。有趣的是，据信仅与5'结构域相互作用的蛋白质S4也能大量且可重复地与颗粒结合。对组装颗粒中RNA的化学探针分析揭示了特征性的切割保护模式，表明这些蛋白质与3'-结构域RNA的组装方式类似于它们与16S rRNA的组装方式，尽管组装的一些后续步骤似乎不完整。这些结果表明，16S rRNA的3'结构域确实可以独立于30S亚基的其余部分组装成一个类似于其在核糖体中结构的颗粒。此外，组装颗粒能够以与30S亚基相当的亲和力结合壮观霉素。
