Ericson G, Chevli K, Wollenzien P
E. A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Medical Center, Missouri 63104.
Biochemistry. 1989 Jul 25;28(15):6446-54. doi: 10.1021/bi00441a043.
16S ribosomal RNA was made by in vitro transcription of a cloned gene, and its structure was compared to authentic 16S ribosomal RNA. The comparison was made by subjecting the two types of 16S rRNA to chemical reagents that react specifically with unpaired bases and determining the extent of reaction by reverse transcription and gel electrophoresis of the cDNA. In solution, the rRNAs were indistinguishable in their pattern of reactivity, except for a difference at A1408 and many differences in the region between residues 470 and 562. When the 16S rRNAs were reconstituted into 30S ribosomal subunits, these reactivity differences were absent. When the synthetic 16S rRNA was reconstituted into 30S subunits and then extracted and tested in solution, its pattern of chemical reactivity in the 470-562 region was the same as authentic 16S rRNA, but differences in the 1408-1410 region persisted. This study indicates that the synthetic 16S rRNA has a secondary structure in solution similar to a native secondary structure except in two regions, one apparently incorrectly folded during synthesis and the other in which nucleotides which are normally methylated in authentic 16S rRNA may be responsible for a structural difference.
16S核糖体RNA通过对克隆基因进行体外转录制备，并将其结构与天然16S核糖体RNA进行比较。比较方法是使这两种类型的16S rRNA与能与未配对碱基特异性反应的化学试剂反应，并通过对cDNA进行逆转录和凝胶电泳来确定反应程度。在溶液中，除了A1408处存在差异以及470至562位残基之间的区域存在许多差异外，这两种rRNA的反应模式无法区分。当将16S rRNA重组到30S核糖体亚基中时，这些反应性差异消失。当将合成的16S rRNA重组到30S亚基中，然后提取并在溶液中进行测试时，其在470 - 562区域的化学反应模式与天然16S rRNA相同，但在1408 - 1410区域的差异仍然存在。这项研究表明，合成的16S rRNA在溶液中的二级结构与天然二级结构相似，除了两个区域，一个区域在合成过程中显然折叠错误，另一个区域中天然16S rRNA中通常被甲基化的核苷酸可能导致了结构差异。
