Effects of site-directed mutations in the central domain of 16 S ribosomal RNA upon ribosomal protein binding, RNA processing and 30 S subunit assembly.
Effects of site-directed mutations in the central domain of 16 S ribosomal RNA upon ribosomal protein binding, RNA processing and 30 S subunit assembly.
复制标题
16 S 核糖体 RNA 中心域的定点突变对核糖体蛋白结合、RNA 加工和 30 S 亚基组装的影响。
DOI：
10.1016/0022-2836(84)90146-3
复制
发表时间：
1984
影响因子：
5.6
通讯作者：
Dahlberg,AE
中科院分区：
文献类型：
--
作者：
Stark,MJ;Gregory,RJ;Gourse,RL;Thurlow,DL;Zwieb,C;Zimmermann,RA;Dahlberg,AE
利用编码大肠杆菌nb核糖体RNA操纵子的多拷贝质粒和镜质体定向诱变技术，我们在16s rRNA的中心区域引入了一些小的改变，其中包含核苷酸560至890。研究的四个rnas包含缺失，一个包含插入。这些改变的小核糖体亚基rnas被用来研究16s rRNA加工、蛋白- 16s rRNA相互作用和30s核糖体亚基组装之间的关系。对大细胞质粒编码转录本的分析表明，野生型16s rRNA基因的产物被完全加工并组装成成熟的30s亚基。在相同条件下，来自突变质粒的转录本的加工和组装严重受损。在某些情况下，突变完全阻断了这两个过程，而在其他情况下，rRNA成熟和核糖体组装被延缓，但没有完全消除。在所有情况下，突变导致17s前体积累为16s rRNA。纯化了突变体的17个S rrna，并与e的不同组合孵育。colirbosomal protein S6, S8， S15和S18，已知它们与16s rRNA的中心结构域结合。核糖核酸酶切得到的蛋白质- 17s rRNA复合物和产物的分离允许检测三种不同的蛋白质- rna片段复合物，包括S8， S8 + S15，或S6 + S8 + S15 + S18。而野生型17s rRNA能够形成所有这三种复合物，核苷酸693至721或822至874的缺失消除了S6和S18的相互作用，核苷酸659至718的去除阻止了S6、S15和S18的结合。相比之下，去除残基614，或在核苷酸614和615之间插入16个碱基，对所测试的四种蛋白质的结合没有显著影响。总之，我们的研究结果表明，16s rRNA成熟和30s亚基组装是紧密耦合的，并且表明，至少在某些情况下，这些过程中的缺陷可能与特定核糖体蛋白无法与改变的rRNA分子相关联有关。此外，我们已经证实了某些rRNA序列对这些蛋白质-rRNA相互作用的形成和/或稳定的重要性。我们的发现也与上一篇文章中对16s rRNA中蛋白质S6、S8、S15和S18结合位点的描述一致，并证明了位点定向诱变作为研究RNA分子中蛋白质结合位点的一种手段的实用性。
Using a multicopy plasmid encoding theEscherichia colirrnB ribosomal RNA operon and the techniques ofin vitrosite-directed mutagenesis, we have introduced several small alterations into the central domain of 16 S rRNA, which encompasses nucleotides 560 to 890. Four of the rRNAs studied contained deletions and one contained an insertion. The altered small ribosomal subunit rRNAs were used to investigate relationships among 16 S rRNA processing, protein- 16 S rRNA interactions and assembly of the 30 S ribosomal subunit. Analysis of plasmid-coded transcripts from maxicells revealed that products from wild-type 16 S rRNA genes were fully processed and assembled into mature 30 S subunits. Under the same conditions, the processing and assembly of transcripts derived from the mutant plasmids were severely impaired. In some instances, the mutations completely blocked both processes, while in other cases rRNA maturation and ribosome assembly were retarded, but not eliminated completely. In all cases, the mutations led to the accumulation of the 17 S precursor to 16 S rRNA. The mutant 17 S rRNAs were purified and incubated with various combinations ofE. coliribosomal proteins S6, S8, S15 and S18, which are known to bind to the central domain of 16 S rRNA. Ribonuclease digestion of the resulting protein-17 S rRNA complexes and fractionation of the products permitted detection of three distinct protein-RNA fragment complexes which contained S8, S8 + S15, or S6 + S8 + S15 + S18. Whereas wild-type 17 S rRNA was able to form all three of these complexes, deletion of nucleotides 693 to 721 or 822 to 874 abolished the interaction of S6 and S18, and removal of nucleotides 659 to 718 prevented the binding of S6, S15 and S18. In contrast, elimination of residue 614, or the presence of a 16-base insertion between nucleotides 614 and 615, had no significant effect on the binding of any of the four proteins tested. Together, our results demonstrate that 16 S rRNA maturation and 30 S subunit assembly are tightly coupled, and show that, in at least some cases, defects in these processes can be correlated with the inability of particular ribosomal proteins to associate with altered rRNA molecules. Moreover, we have confirmed the essentiality of certain rRNA sequences for the formation and/or stabilization of these protein-rRNA interactions. Our findings are also consistent with the description of the binding sites for proteins S6, S8, S15 and S18 in the 16 S rRNA that was presented in the preceding paper and demonstrate the utility of sitedirected mutagenesis as a means for studying protein binding sites in RNA molecules.