Cheung J, Lin A
Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan, ROC.
J Biomed Sci. 1999 Jul-Aug;6(4):277-84. doi: 10.1007/BF02253569.
The regional structure of the sarcin domain of 23S rRNA of Escherichia coli ribosomes was determined by a combinatory approach of oligo DNA probes and the action of alpha-sarcin. The sarcin domain is protected by a reactive complementary oligo DNA probe against the hydrolytic action of alpha-sarcin. This protective effect is dependent upon the length and the complementary sequence of oligo DNA probes that react to ribosomes. Under UV irradiation and using of the primer extension, nucleotides that contacted by reactive oligo DNA probes were determined. Nucleotides at the 3' side of the domain (positions from G2659 to C2676) were targeted by oligo DNA probes that have their sequences to complement the domain, indicating that the 3' side region was exposed on the surface of ribosomes, whereas nucleotides at the 5' side of stem and extented to two bases at the loop (positions from C2646 to A2654) were not accessible to any oligo DNA probes, implying that the region could be buried in ribosomes. This study also provided evidence that the conformation of the sarcin domain is subjected to alteration if the exposed 3' side of domain is targeted by the reactive DNA probe. The importance of the topological arrangement of the sarcin domain that engages in the translocation event during translation is discussed.
通过寡聚DNA探针与α-肌动蛋白作用的组合方法，确定了大肠杆菌核糖体23S rRNA肌动蛋白结构域的区域结构。肌动蛋白结构域受到反应性互补寡聚DNA探针的保护，免受α-肌动蛋白的水解作用。这种保护作用取决于与核糖体反应的寡聚DNA探针的长度和互补序列。在紫外线照射下并使用引物延伸法，确定了与反应性寡聚DNA探针接触的核苷酸。该结构域3'端（从G2659到C2676的位置）的核苷酸被具有与该结构域互补序列的寡聚DNA探针靶向，表明3'端区域暴露在核糖体表面，而茎部5'端并延伸到环上两个碱基（从C2646到A2654的位置）的核苷酸对任何寡聚DNA探针都不可接近，这意味着该区域可能埋在核糖体中。这项研究还提供了证据，即如果该结构域暴露的3'端被反应性DNA探针靶向，肌动蛋白结构域的构象会发生改变。讨论了在翻译过程中参与转位事件的肌动蛋白结构域拓扑排列的重要性。
