Pardon B, Thelen L, Wagner R
Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Germany.
Biol Chem Hoppe Seyler. 1994 Jan;375(1):11-20. doi: 10.1515/bchm3.1994.375.1.11.
The structure of the E. coli ribosomal RNA leader was analyzed by treatment with single and double strand specific ribonucleases and by chemical modification. The experimentally derived data together with secondary structure calculations according to minimum free energy was used to construct a secondary structure model. The binding of purified Nus proteins to the ribosomal leader RNA was further tested. Contrary to the recently reported interactions of NusB and NusE with a nut RNA sequence we obtained evidence that the presence of NusA and NusE resulted in protection against hydroxyl radical reaction of the leader nut elements boxA, boxB and boxC. The possible significance of this interaction is discussed. In the second part of the study we analyzed effects of leader mutations, which are known to affect cell growth, on the activity of ribosomes in vivo. A system was used able to distinguish the proportion of ribosomes assembled from rRNA of chromosomal origin (wild type) and plasmid origin (mutant). It turned out that the amount of 16S RNA transcribed from genes with point mutations in the leader region decreased if ribosomal pools with different translational activities were compared. High amounts of transcripts from mutant operons were present in the free ribosomal RNA and the free 30S fractions. Significantly less 16S RNA transcripts from the mutated genes were detected in the functionally active and homogeneous 70S tight couple preparations, and even less in the polysome fraction involved in active translation. The results allow a better understanding of the function of rRNA leader sequences in structure formation and correct ribosome biogenesis.
通过用单链和双链特异性核糖核酸酶处理以及化学修饰，对大肠杆菌核糖体RNA前导序列的结构进行了分析。将实验获得的数据与根据最小自由能进行的二级结构计算相结合，用于构建二级结构模型。进一步测试了纯化的Nus蛋白与核糖体前导RNA的结合。与最近报道的NusB和NusE与nut RNA序列的相互作用相反，我们获得的证据表明，NusA和NusE的存在导致前导nut元件boxA、boxB和boxC对羟基自由基反应具有抗性。讨论了这种相互作用的可能意义。在研究的第二部分中，我们分析了已知会影响细胞生长的前导序列突变对体内核糖体活性的影响。使用了一种系统，能够区分由染色体来源（野生型）和质粒来源（突变型）的rRNA组装而成的核糖体比例。结果表明，比较具有不同翻译活性的核糖体库时，前导区域存在点突变的基因转录的16S RNA量会减少。来自突变操纵子的大量转录本存在于游离核糖体RNA和游离30S组分中。在功能活性且均一的70S紧密偶联制剂中检测到的来自突变基因的16S RNA转录本明显较少，而在参与活跃翻译的多核糖体组分中更少。这些结果有助于更好地理解rRNA前导序列在结构形成和正确核糖体生物发生中的功能。
