Zaporojets Dmitry, French Sarah, Squires Catherine L
Department of Infectious Diseases, Boston Medical Center, Boston, Massachusetts 02118, USA.
J Bacteriol. 2003 Dec;185(23):6921-7. doi: 10.1128/JB.185.23.6921-6927.2003.
To examine the flexibility of rRNA operons with respect to fundamental organization, transcription, processing, and assembly of ribosomes, operon variations were introduced by a plasmid into an Escherichia coli strain that has deletions of all chromosomal copies of rRNA genes. In the reconstructed operons, a Salmonella intervening sequence (IVS) from 23S helix 45 was introduced into the E. coli 23S gene at the same position. Three different constructs of the E. coli 16S gene were then placed wholly within the IVS sequence, and the 16S gene was deleted from its normal position. The resulting plasmids thus had the normal operon promoters and the leader region followed by the 5' one-third of the 23S gene, the entire 16S gene within the IVS, the last two-thirds of the 23S gene, and the normal end of the operon. The three constructs differed in the amount of 16S leader and spacer regions they contained. Only two of the three constructs, those with redundant leader and spacer antiterminator signals, resulted in viable cultures of the rrn deletion strain. Electron micrographs of the variant operon suggest that the 23S rRNA is made in two separate parts which then must form subassemblies before assembling into a functional 50S subunit. Cells containing only the reshuffled genes were debilitated in their growth properties and ribosome contents. The fact that such out of the ordinary manipulation of rRNA sequences in E. coli is possible paves the way for detailed analysis of ribosome assembly and evolution.
为了研究核糖体RNA操纵子在核糖体基本组织、转录、加工和组装方面的灵活性，通过质粒将操纵子变异引入到一个缺失了所有rRNA基因染色体拷贝的大肠杆菌菌株中。在重建的操纵子中，将来自23S螺旋45的沙门氏菌间隔序列（IVS）引入到大肠杆菌23S基因的相同位置。然后将三种不同构建的大肠杆菌16S基因完全置于IVS序列内，并将16S基因从其正常位置删除。由此产生的质粒因此具有正常的操纵子启动子和前导区域，随后是23S基因的5'三分之一、IVS内的整个16S基因、23S基因的最后三分之二以及操纵子的正常末端。这三种构建体在它们所含的16S前导和间隔区域的数量上有所不同。三种构建体中只有两种，即具有冗余前导和间隔抗终止信号的构建体，导致了rrn缺失菌株的活培养物。变异操纵子的电子显微镜照片表明，23S rRNA是分两个独立部分合成的，然后在组装成功能性50S亚基之前必须形成亚组装体。仅含有重新排列基因的细胞在其生长特性和核糖体含量方面有所减弱。在大肠杆菌中对rRNA序列进行这种非同寻常的操作是可能的，这一事实为核糖体组装和进化的详细分析铺平了道路。
