RAPID CHEMICAL PROBING OF CONFORMATION IN 16-S RIBOSOMAL-RNA AND 30-S RIBOSOMAL-SUBUNITS USING PRIMER EXTENSION
RAPID CHEMICAL PROBING OF CONFORMATION IN 16-S RIBOSOMAL-RNA AND 30-S RIBOSOMAL-SUBUNITS USING PRIMER EXTENSION
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DOI：
10.1016/0022-2836(86)90441-9
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发表时间：
1986-02-05
影响因子：
5.6
通讯作者：
NOLLER, HF
中科院分区：
文献类型：
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作者：
MOAZED, D;STERN, S;NOLLER, HF
我们已经详细研究了16 S核糖体RNA的高级结构，无论是在其裸露的形式和30 S核糖体亚基。使用乙二醛（在N1和N2处与鸟嘌呤反应）、硫酸二甲酯（在N1处与腺嘌呤反应，在N3处与胞嘧啶反应）和1-环己基-3-（2-吗啉代乙基）-碳二亚胺甲对甲苯磺酸盐（在N3处与尿嘧啶反应，在N1处与鸟嘌呤反应）探测16 S rRNA链中的每个碱基。通过使用合成的寡脱氧核苷酸引物用逆转录酶进行引物延伸来鉴定反应位点。这些结果提供了一个详细的和严格的实验测试模型的16 S rRNA二级结构，这是来自主要的比较序列分析。我们的数据还提供了有关16 S rRNA的三级和四级结构的信息。与裸16 S rRNA获得的数据显示合理的接近协议与建议的模型，并与30 S亚基获得的数据显示几乎完全一致。 除了明显的整体结构“收紧”（其中许多弱反应性碱基变得不反应）外，蛋白质与16 S rRNA组装形成30 S亚基带来了许多局部结构重排，导致特异性增强和保护。在许多情况下，核糖体蛋白似乎“调整”16 S rRNA结构，使其符合遗传学预测的模型，即使RNA本身似乎经常在分子的某些区域偏好不同的结构。保守的，未配对的腺嘌呤形成30 S亚基后，广泛的保护表明，他们在组装过程中发挥了特殊的作用，可能提供信号的蛋白质识别。
We have investigated in detail the higher-order structure of 16 S ribosomal RNA, both in its naked form and in 30 S ribosomal subunits. Each base in the 16 S rRNA chain has been probed using kethoxal (which reacts with guanine at N1 and N2), dimethylsulfate (which reacts with adenine at N1 and cytosine at N3) and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate (which reacts with uracil at N3 and guanine at N1). The sites of reaction were identified by primer extension with reverse transcriptase using synthetic oligodeoxynucleotide primers. These results provide a detailed and rigorous experimental test of a model for 16 S rRNA secondary structure, which was derived mainly from comparative sequence analysis. Our data also provide information relevant to tertiary and quaternary structure of 16 S rRNA. Data obtained with naked 16 S rRNA show reasonably close agreement with the proposed model, and data obtained with 30 S subunits show nearly complete agreement. Apart from an apparent overall "tightening" of the structure (in which many weakly reactive bases become unreactive), assembly of the proteins with 16 S rRNA to form 30 S subunits brings about numerous local structural rearrangements, resulting in specific enhancements as well as protections. In many instances, the ribosomal proteins appear to "tune" the 16 S rRNA structure to bring it into accordance with the phylogenetically predicted model, even though the RNA on its own often seems to prefer a different structure in certain regions of the molecule. Extensive protection of conserved, unpaired adenines upon formation of 30 S subunits suggests that they play a special role in the assembly process, possibly providing signals for protein recognition.