Malhotra A, Harvey S C
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham 35294.
J Mol Biol. 1994 Jul 22;240(4):308-40. doi: 10.1006/jmbi.1994.1448.
We use a computer-based protocol for automated structure refinement of large RNAs and ribonucleoproteins to propose a three-dimensional model for the Escherichia coli 16 S RNA in the 30 S ribosomal subunit along with the first quantitative estimates of the uncertainties in the model. Our models are based on the 16 S RNA secondary structure, the small angle neutron scatter map of 30 S proteins, tertiary RNA-RNA and RNA-protein contacts as suggested by cross-linking, chemical footprinting and other experimental studies, and electron microscopy data for the shape of the 30 S subunit and placement of 16 S RNA fragments, along with known motifs in RNA structure. In addition, some data on the interaction of the tRNAs/mRNA with the 16 S RNA were used to localize the active site. Since there are not enough structural data to derive a unique three-dimensional folding of the 16 S RNA, several different conformations can be generated to satisfy the experimental data. A set of seven models was refined to survey the range of acceptable conformations. These models were analyzed to deduce probable positions and orientations of the different helical segments that comprise the 16 S RNA in the Escherichia coli small subunit, and one consensus model from this set is presented here. An estimate of the reliability of our predicted structure is made using the variations between the models, and about 75% of 16 S RNA helical segments are localized to 15 A or less in their position in the small subunit. Our models show a distinct separation of the three major domains of the 16 S RNA. The 5' major domain and the central domain are clustered in the body of the 30 S subunit, whereas the 3' major domain is localized in the head of the subunit. Our modeling results are compared with models of the 16 S RNA proposed by other researchers, and are seen to be similar to the manually built models by Stern et al. and Brimacombe et al. with a few significant differences. The position of nucleotides implicated by footprinting and crosslinking data in tRNA and mRNA binding, and in subunit association are examined, and many of these sites are seen to lie along the 30 S subunit neck, cleft and the platform.
我们使用基于计算机的协议对大型RNA和核糖核蛋白进行自动结构优化，以提出大肠杆菌30S核糖体亚基中16S RNA的三维模型，并首次对模型中的不确定性进行了定量估计。我们的模型基于16S RNA二级结构、30S蛋白的小角中子散射图、交联、化学足迹和其他实验研究表明的三级RNA-RNA和RNA-蛋白相互作用，以及30S亚基形状和16S RNA片段位置的电子显微镜数据，以及RNA结构中的已知基序。此外，还使用了一些关于tRNA/mRNA与16S RNA相互作用的数据来定位活性位点。由于没有足够的结构数据来推导16S RNA的独特三维折叠，因此可以生成几种不同的构象来满足实验数据。我们优化了一组七个模型，以研究可接受构象的范围。对这些模型进行分析，以推断大肠杆菌小亚基中构成16S RNA的不同螺旋片段的可能位置和方向，本文给出了该组中的一个共识模型。利用模型之间的差异对我们预测结构的可靠性进行了估计，在小亚基中，约75%的16S RNA螺旋片段定位在15埃或更小的范围内。我们的模型显示了16S RNA三个主要结构域的明显分离。5'主要结构域和中央结构域聚集在30S亚基的主体中，而3'主要结构域位于亚基的头部。我们将建模结果与其他研究人员提出的16S RNA模型进行了比较，发现与Stern等人和Brimacombe等人手工构建的模型相似，但也有一些显著差异。我们研究了足迹和交联数据所涉及的tRNA和mRNA结合以及亚基缔合中核苷酸的位置，发现其中许多位点位于30S亚基的颈部、裂隙和平台上。
