Brimacombe R, Atmadja J, Stiege W, Schüler D
Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin-Dahlem, Germany.
J Mol Biol. 1988 Jan 5;199(1):115-36. doi: 10.1016/0022-2836(88)90383-x.
A large body of intra-RNA and RNA-protein crosslinking data, obtained in this laboratory, was used to fold the phylogenetically and experimentally established secondary structure of Escherichia coli 16 S RNA into a three-dimensional model. All the crosslinks were induced in intact 30 S subunits (or in some cases in growing E. coli cells), and the sites of crosslinking were precisely localized on the RNA by oligonucleotide analysis. The RNA-protein crosslinking data (including 28 sites, and involving 13 of the 21 30S ribosomal were used to relate the RNA structure to the distribution of the proteins as determined by neutron scattering. The three-dimensional model of the 16 S RNA has overall dimensions of 220 A x 140 A x 90 A, in good agreement with electron microscopic estimates for the 30 S subunit. The shape of the model is also recognizably the same as that seen in electron micrographs, and the positions in the model of bases localized on the 30 S subunit by immunoelectron microscopy (the 5' and 3' termini, the m7G and m6(2)A residues, and C-1400) correspond closely to their experimentally observed positions. The distances between the RNA-protein crosslink sites in the model correlate well with the distances between protein centres of mass obtained by neutron scattering, only two out of 66 distances falling outside the expected tolerance limits. These two distances both involve protein S13, a protein noted for its anomalous behaviour. A comparison with other experimental information not specifically used in deriving the model shows that it fits well with published data on RNA-protein binding sites, mutation sites on the RNA causing resistance to antibiotics, tertiary interactions in the RNA, and a potential secondary structural "switch". Of the sites on 16 S RNA that have been found to be accessible to chemical modification in the 30 S subunit, 87% are at obviously exposed positions in the model. In contrast, 70% of the sites corresponding to positions that have ribose 2'-O-methylations in the eukaryotic 18 S RNA from Xenopus laevis are at non-exposed (i.e. internal) positions in the model. All nine of the modified bases in the E. coli 16 S RNA itself show a remarkable distribution, in that they form a "necklace" in one plane around the "throat" of the subunit. Insertions in eukaryotic 18 S RNA, and corresponding deletions in chloroplast or mammalian mitochondrial ribosomal RNA relative to E. coli 16 S RNA represent distinct sub-domains in the structure.(ABSTRACT TRUNCATED AT 400 WORDS)
本实验室获得的大量RNA内部及RNA-蛋白质交联数据，被用于将通过系统发育和实验确定的大肠杆菌16S RNA二级结构折叠成三维模型。所有交联都是在完整的30S亚基中诱导产生的（在某些情况下是在生长的大肠杆菌细胞中），并且通过寡核苷酸分析将交联位点精确地定位在RNA上。RNA-蛋白质交联数据（包括28个位点，涉及21种30S核糖体蛋白中的13种）被用于将RNA结构与通过中子散射确定的蛋白质分布联系起来。16S RNA的三维模型总体尺寸为220Å×140Å×90Å，与30S亚基的电子显微镜估计值高度一致。该模型的形状也与电子显微镜照片中看到的明显相同，并且通过免疫电子显微镜定位在30S亚基上的碱基在模型中的位置（5'和3'末端、m7G和m6(2)A残基以及C-1400）与它们的实验观察位置密切对应。模型中RNA-蛋白质交联位点之间的距离与通过中子散射获得的蛋白质质心之间的距离相关性良好，66个距离中只有两个超出预期的公差范围。这两个距离都涉及蛋白质S13，该蛋白质因其异常行为而受到关注。与在推导模型时未专门使用的其他实验信息进行比较表明，它与已发表的关于RNA-蛋白质结合位点、导致抗生素抗性的RNA突变位点、RNA中的三级相互作用以及潜在的二级结构“开关”的数据非常吻合。在30S亚基中已发现可进行化学修饰的16S RNA位点中，87%在模型中处于明显暴露的位置。相比之下，非洲爪蟾真核18S RNA中对应于具有核糖2'-O-甲基化位置的位点，70%在模型中处于非暴露（即内部）位置。大肠杆菌16S RNA本身的所有九个修饰碱基呈现出显著的分布，即它们在围绕亚基“喉部”的一个平面上形成一条“项链”。真核18S RNA中的插入以及相对于大肠杆菌16S RNA在叶绿体或哺乳动物线粒体核糖体RNA中的相应缺失代表了结构中的不同子域。（摘要截断于400字）
