Conrad C, Rauhut R, Klug G
Institut für Mikro- und Molekularbiologie der Justus-Liebig-Universität Giessen, Frankfurter Strasse 107,35392 Giessen, Germany.
Nucleic Acids Res. 1998 Oct 1;26(19):4446-53. doi: 10.1093/nar/26.19.4446.
23S rRNA in Rhodobacter capsulatus shows endoribonuclease III (RNase III)-dependent fragmentation in vivo at a unique extra stem-loop extending from position 1271 to 1331. RNase III is a double strand (ds)-specific endoribonuclease. This substrate preference is mediated by a double-stranded RNA binding domain (dsRBD) within the protein. Although a certain degree of double strandedness is a prerequisite, the question arises what structural features exactly make this extra stem-loop an RNase III cleavage site, distinguishing it from the plethora of stem-loops in 23S rRNA? We used RNase III purified from R.capsulatus and Escherichia coli, respectively, together with well known substrates for E.coli RNase III and RNA substrates derived from the special cleavage site in R.capsulatus 23S rRNA to study the interaction between the Rhodobacter enzyme and the fragmentation site. Although both enzymes are very similar in their amino acid sequence, they exhibit significant differences in binding and cleavage of these in vitro substrates.
荚膜红细菌中的23S rRNA在体内会在一个从1271位延伸至1331位的独特额外茎环处发生依赖核糖核酸酶III（RNase III）的片段化。RNase III是一种双链（ds）特异性核糖核酸内切酶。这种底物偏好性由该蛋白质内的一个双链RNA结合结构域（dsRBD）介导。尽管一定程度的双链性是一个先决条件，但问题是究竟哪些结构特征使得这个额外的茎环成为RNase III的切割位点，从而将其与23S rRNA中大量的茎环区分开来？我们分别使用从荚膜红细菌和大肠杆菌中纯化的RNase III，以及大肠杆菌RNase III的已知底物和源自荚膜红细菌23S rRNA特殊切割位点的RNA底物，来研究红细菌酶与片段化位点之间的相互作用。尽管这两种酶在氨基酸序列上非常相似，但它们在这些体外底物的结合和切割方面表现出显著差异。
