Gravel M, Leclerc D, Melançon P, Brakier-Gingras L
Département de Biochimie, Université de Montréal, Canada.
Nucleic Acids Res. 1989 Apr 11;17(7):2723-32. doi: 10.1093/nar/17.7.2723.
Plasmid pPM114 carries the Escherichia coli 16S ribosomal RNA gene under the control of a T7 promoter. It can generate in vitro transcribed 16S rRNA that can be assembled into functional 30S ribosomal subunits. Two deletion mutants were derived from pPM114, by partial or total deletion of the conserved 900 stem/loop region of the 16S rRNA. These mutants, pMG delta 10 and pMG delta 23, respectively lack bases 895 to 904 and 889 to 911 of the 16S rRNA. The amputated 16S rRNA transcripts synthesized from these mutated plasmids were assembled into 30S subunits which were as active under the direction of an artificial or a natural messenger as subunits reconstructed with the full-length 16S rRNA transcript. They also responded as well to the stimulation of misreading by streptomycin, although the deleted region is proximal to the streptomycin binding domain. However, when we attempted to delete the 895-904 or 889-911 region from the 16S rRNA gene in plasmid pKK3535 which carries the rrnB operon, no transformants harbouring plasmids with one of these deletions could be recovered. These observations suggest that the 900 stem/loop region of the 16S rRNA is not required for the ribosomal function but is probably essential for important cell regulatory functions.
质粒pPM114携带受T7启动子控制的大肠杆菌16S核糖体RNA基因。它可以产生体外转录的16S rRNA，后者能够组装成功能性的30S核糖体亚基。通过部分或完全缺失16S rRNA保守的900茎环区域，从pPM114衍生出两个缺失突变体。这些突变体pMG delta 10和pMG delta 23分别缺失16S rRNA的895至904位碱基和889至911位碱基。从这些突变质粒合成的截短16S rRNA转录本被组装成30S亚基，在人工或天然信使的指导下，其活性与用全长16S rRNA转录本重建的亚基相同。它们对链霉素引起的错读刺激也有同样的反应，尽管缺失区域靠近链霉素结合结构域。然而，当我们试图从携带rrnB操纵子的质粒pKK3535的16S rRNA基因中删除895 - 904或889 - 911区域时，未能获得携带这些缺失之一的质粒的转化体。这些观察结果表明，16S rRNA的900茎环区域对于核糖体功能不是必需的，但可能对重要的细胞调节功能至关重要。
