Theissen G, Thelen L, Wagner R
Heinrich-Heine-Universität Düsseldorf Institut für Physikalische Biologie, Germany.
J Mol Biol. 1993 Sep 20;233(2):203-18. doi: 10.1006/jmbi.1993.1500.
Recently, we reported on the creation of a systematic series of C to T transition mutations, located between 19 and 45 nucleotides upstream of the mature 16 S RNA 5' end of a complete, plasmid encoded ribosomal RNA operon. We showed that some of these base transitions have pronounced effects on the growth phenotype of mutant cells, and on the stability of their 16 S RNA as well as on the association capability of their ribosomal subunits. From these observations we concluded that the mutated leader region has post-transcriptional functions in the biogenesis of ribosomes. To further substantiate our conclusions we have now analyzed the growth phenotypes of some leader mutants in more detail, and show here that they are temperature dependent. Furthermore, we have isolated ribosomal RNA, 70 S ribosomes and ribosomal subunits from wild-type and mutant strains and subjected them to a detailed structural and functional analysis. We show that processing and maturation of the 16 S RNA is not altered as a consequence of the base transitions in the leader. In contrast, comparison of the protein composition of wild-type and mutant 30 S particles by two-dimensional gel electrophoresis revealed specific differences. Wild-type 30 S subunits, which are not tightly bound to 50 S, are lacking many ribosomal proteins, while the same fraction of ribosomes from mutant cells has an approximately complete r-protein set, and instead contains some additional non-ribosomal proteins. The translational activity of mutant and wild-type total ribosome preparations was analyzed in vitro. Ribosomes from slowly growing mutants show a significantly reduced in vitro translational activity, which is caused by the 30 S subunits. In contrast, the defects in association reside mainly in the 50 S subunits. Our results demonstrate that some base substitutions in the leader of an Escherichia coli rRNA operon affect the structure and function of ribosomes, although the mutated region is not part of the particles finally formed. This finding implies that at least part of the leader region assists the structure formation of functional 30 S subunits, before it is cut away and discarded. We argue that the rrn leader thus fulfills the functional criteria of a transient molecular scaffold.
最近，我们报道了一系列系统性的C到T转换突变的产生，这些突变位于一个完整的、质粒编码的核糖体RNA操纵子成熟16S RNA 5'端上游19至45个核苷酸之间。我们发现其中一些碱基转换对突变细胞的生长表型、其16S RNA的稳定性以及核糖体亚基的结合能力有显著影响。基于这些观察结果，我们得出结论，突变的前导区域在核糖体生物合成中具有转录后功能。为了进一步证实我们的结论，我们现在更详细地分析了一些前导突变体的生长表型，并在此表明它们是温度依赖性的。此外，我们从野生型和突变菌株中分离出核糖体RNA、70S核糖体和核糖体亚基，并对它们进行了详细的结构和功能分析。我们发现前导区域的碱基转换不会改变16S RNA的加工和成熟过程。相反，通过二维凝胶电泳比较野生型和突变型30S颗粒的蛋白质组成，发现了特定差异。未与50S紧密结合的野生型30S亚基缺少许多核糖体蛋白，而来自突变细胞的相同核糖体部分具有大致完整的核糖体蛋白集，并且还包含一些额外的非核糖体蛋白。对突变型和野生型总核糖体制剂的体外翻译活性进行了分析。来自生长缓慢的突变体的核糖体显示出体外翻译活性显著降低，这是由30S亚基引起的。相比之下，结合缺陷主要存在于50S亚基中。我们的结果表明，大肠杆菌rRNA操纵子前导区域的一些碱基替换会影响核糖体的结构和功能，尽管突变区域不是最终形成的颗粒的一部分。这一发现意味着至少部分前导区域在被切除和丢弃之前有助于功能性30S亚基的结构形成。我们认为rrn前导序列因此符合瞬时分子支架的功能标准。
