Li Z, Pandit S, Deutscher M P
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101, USA.
RNA. 1999 Jan;5(1):139-46. doi: 10.1017/s1355838299981669.
Ribosomal RNAs are generally synthesized as long, primary transcripts that must be extensively processed to generate the mature, functional species. In Escherichia coli, it is known that the initial 30S precursor is cleaved during its synthesis by the endonuclease RNase III to generate precursors to the 16S, 23S, and 5S rRNAs. However, despite extensive study, the processes by which these intermediate products are converted to their mature forms are poorly understood. In this article, we describe the maturation of 23S rRNA. Based on Northern analysis of RNA isolated from a variety of mutant strains lacking one or multiple ribonucleases, we show that maturation of the 3' terminus requires the action of RNase T, an enzyme previously implicated in the end turnover of tRNA and in the maturation of small, stable RNAs. Although other exoribonucleases can participate in shortening the 3' end of the initial RNase III cleavage product, RNase T is required for removal of the last few residues. In the absence of RNase T, 23S rRNA products with extra 3' residues accumulate and are incorporated into ribosomes, with only small effects on cell growth. Purified RNase T accurately and efficiently converts these immature ribosomes to their mature forms in vitro, whereas free RNA is processed relatively poorly. In vivo, the processing defect at the 3' end has no effect on 5' maturation, indicating that the latter process proceeds independently. We also find that a portion of the 23S rRNA that accumulates in many RNase T- cells becomes polyadenylated because of the action of poly(A) polymerase I. The requirement for RNase T in 23S rRNA maturation is discussed in relation to a model in which only this enzyme, among the eight exoribonucleases present in E. coli, is able to efficiently remove nucleotides close to the double-stranded stem generated by the pairing of the 5' and 3' termini of most stable RNAs.
核糖体RNA通常作为长的初级转录本进行合成，这些转录本必须经过广泛加工才能产生成熟的、具有功能的产物。在大肠杆菌中，已知最初的30S前体在其合成过程中被内切核酸酶RNase III切割，以产生16S、23S和5S rRNA的前体。然而，尽管进行了广泛研究，但这些中间产物转化为成熟形式的过程仍知之甚少。在本文中，我们描述了23S rRNA的成熟过程。基于对从多种缺乏一种或多种核糖核酸酶的突变菌株中分离的RNA进行的Northern分析，我们表明3'末端的成熟需要RNase T的作用，该酶先前与tRNA的末端周转以及小的稳定RNA的成熟有关。尽管其他外切核糖核酸酶可以参与缩短最初RNase III切割产物的3'末端，但去除最后几个残基需要RNase T。在没有RNase T的情况下，带有额外3'残基的23S rRNA产物会积累并被整合到核糖体中，对细胞生长只有很小的影响。纯化的RNase T在体外能准确高效地将这些未成熟的核糖体转化为成熟形式，而游离RNA的加工效果相对较差。在体内，3'末端的加工缺陷对5'成熟没有影响，这表明后者过程是独立进行的。我们还发现，在许多缺乏RNase T的细胞中积累的一部分23S rRNA由于聚腺苷酸聚合酶I的作用而被聚腺苷酸化。结合一个模型讨论了23S rRNA成熟过程中对RNase T的需求，在该模型中，在大肠杆菌中存在的八种外切核糖核酸酶中，只有这种酶能够有效去除靠近大多数稳定RNA的5'和3'末端配对产生的双链茎的核苷酸。
