Lee K, Holland-Staley C A, Cunningham P R
Department of Biological Sciences, Wayne State University, Detroit, MI 48202, USA.
J Nutr. 2001 Nov;131(11):2994S-3004S. doi: 10.1093/jn/131.11.2994S.
A genetic system for the study of ribosomal RNA function and structure was developed. First, the ribosome binding sequence of the chloramphenicol acetyltransferase gene and the message binding sequence of 16S ribosomal RNA were randomly mutated and alternative highly functional sequences were selected and characterized. From this set of mutants, a single clone was chosen and subjected to a second round of mutagenesis to optimize the specificity of the system. In the resulting system, plasmid-encoded ribosomes efficiently and exclusively translate specific mRNA containing the appropriate ribosome binding sequences. This system allows facile isolation and analysis of mutations that would normally be lethal and allows direct selection of rRNA mutants with predetermined levels of ribosome function. The system was used to examine the effects of mutations at the sole pseudouridine (Psi) in Escherichia coli 16S rRNA which is located at position 516 of the conserved 530 loop. The nucleotide opposite Psi516 in the hairpin, A535, was also mutated. The data show that a pyrimidine (Psi or C) is required at position 516, while substitutions at position 535 reduce ribosome function by < 50%. A requirement for base pair formation between Psi516 and A535 was not indicated.
开发了一种用于研究核糖体RNA功能和结构的遗传系统。首先，氯霉素乙酰转移酶基因的核糖体结合序列和16S核糖体RNA的信使结合序列被随机突变，然后选择并鉴定出替代的高功能序列。从这组突变体中挑选出一个克隆，并进行第二轮诱变以优化该系统的特异性。在所得系统中，质粒编码的核糖体高效且专一性地翻译含有适当核糖体结合序列的特定mRNA。该系统便于分离和分析通常会致死的突变，并允许直接选择具有预定核糖体功能水平的rRNA突变体。该系统用于研究大肠杆菌16S rRNA中位于保守530环516位的唯一假尿苷（Ψ）处的突变效应。发夹结构中与Ψ516相对的核苷酸A535也发生了突变。数据表明，516位需要一个嘧啶（Ψ或C），而535位的取代使核糖体功能降低不到50%。未表明Ψ516和A535之间需要形成碱基对。
