Poot R A, Brink M F, Pleij C W, de Boer H A, van Duin J
Department of Biochemistry, Gorlaeus Laboratories, University of Leiden, The Netherlands.
Nucleic Acids Res. 1993 Nov 25;21(23):5398-402. doi: 10.1093/nar/21.23.5398.
We describe a system to isolate 30S ribosomal subunits which contain targeted mutations in their 16S rRNA. The mutations of interest should be present in so-called specialized 30S subunits which have an anti-Shine-Dalgarno sequence that is altered from 5' ACCUCC to 5' ACACAC. These plasmid-encoded specialized 30S subunits are separated from their chromosomally encoded wild-type counterparts by affinity chromatography that exploits the different Shine-Dalgarno complementarity. An oligonucleotide complementary to the 3' end of wild-type 16S rRNA and attached to a solid phase matrix retains the wild-type 30S subunits. The flow-through of the column contains close to 100% mutant 30S subunits. Toeprinting assays demonstrate that affinity column treatment does not cause significant loss of activity of the specialized particles in initiation complex formation, whereas elongation capacity as determined by poly(Phe) synthesis is only slightly decreased. The method described offers an advantage over total reconstitution from in vitro transcribed mutant 16S rRNA since our 30S subunits contain the naturally occurring base modifications in their 16S rRNA.
我们描述了一种用于分离30S核糖体亚基的系统，这些亚基在其16S rRNA中含有靶向突变。感兴趣的突变应存在于所谓的特殊30S亚基中，这些亚基具有从5' ACCUCC改变为5' ACACAC的反Shine-Dalgarno序列。这些质粒编码的特殊30S亚基通过利用不同的Shine-Dalgarno互补性的亲和色谱法与它们染色体编码的野生型对应物分离。与野生型16S rRNA的3'末端互补并连接到固相基质上的寡核苷酸保留野生型30S亚基。柱的流出物含有接近100%的突变30S亚基。足迹分析表明，亲和柱处理不会导致特殊颗粒在起始复合物形成中的活性显著丧失，而由聚（苯丙氨酸）合成测定的延伸能力仅略有下降。所述方法相对于从体外转录的突变16S rRNA进行完全重建具有优势，因为我们的30S亚基在其16S rRNA中含有天然存在的碱基修饰。
