Merryman C, Moazed D, Daubresse G, Noller H F
Center for Molecular Biology of RNA, Sinsheimer Laboratories, Santa Cruz, CA, 95064, USA.
J Mol Biol. 1999 Jan 8;285(1):107-13. doi: 10.1006/jmbi.1998.2243.
We have studied the effect of subunit association on the accessibility of nucleotides in 23S and 5S rRNA. Escherichia coli 50S subunits and 70S ribosomes were subjected to a combination of chemical probes and the sites of attack identified by primer extension. Since the ribose groups and all of the bases were probed, the present study provides a comprehensive map of the nucleotides that are likely to be involved in subunit-subunit interactions. Upon subunit association, the bases of 22 nucleotides and the ribose groups of more than 60 nucleotides are protected in 23S rRNA; no changes are seen in 5S rRNA. Interestingly, the bases of nucleotides A1866, A1891 and A1896, and G2505 become more reactive to chemical probes, indicating localized rearrangement of the structure of the 50S subunit upon association with the 30S subunit. Most of the protected nucleotides are located in four stem-loop structures around positions 715, 890, 1700, and 1920. In free 50S subunits, virtually all of the ribose groups in these four regions are strongly cleaved by hydroxyl radicals, suggesting that these stems protrude from the 50S subunit. When the 30S subunit is bound, most of the ribose groups in the 715, 890, 1700 and 1920 stem-loops are protected, as are many bases in and around the corresponding apical loops. Intriguingly, three of the protected regions of 23S rRNA are known to be linked via tertiary interactions to features of the peptidyl transferase center. Together with the juxtaposition of the subunit-protected regions of 16S rRNA with the small subunit tRNA binding sites, our findings suggest the existence of a communication pathway between the codon-anticodon binding sites of the 30S subunit with the peptidyl transferase center of the 50S subunit via rRNA-rRNA interactions.
我们研究了亚基缔合对23S和5S核糖体RNA中核苷酸可及性的影响。对大肠杆菌50S亚基和70S核糖体进行了化学探针组合处理，并通过引物延伸确定攻击位点。由于核糖基团和所有碱基都被探测，本研究提供了可能参与亚基-亚基相互作用的核苷酸的全面图谱。亚基缔合后，23S核糖体RNA中22个核苷酸的碱基和60多个核苷酸的核糖基团受到保护；5S核糖体RNA中未见变化。有趣的是，核苷酸A1866、A1891和A1896以及G2505的碱基对化学探针的反应性增强，表明50S亚基与30S亚基缔合时结构发生局部重排。大多数受保护的核苷酸位于715、890、1700和1920位附近的四个茎环结构中。在游离的50S亚基中，这四个区域的几乎所有核糖基团都被羟基自由基强烈切割，表明这些茎从50S亚基突出。当结合30S亚基时，715、890、1700和1920茎环中的大多数核糖基团受到保护，相应顶端环内及周围的许多碱基也受到保护。有趣的是，已知23S核糖体RNA的三个受保护区域通过三级相互作用与肽基转移酶中心的特征相连。结合16S核糖体RNA的亚基保护区域与小亚基tRNA结合位点的并列，我们的发现表明30S亚基的密码子-反密码子结合位点与50S亚基的肽基转移酶中心之间通过rRNA-rRNA相互作用存在一条通信途径。
