Amara R R, Vijaya S
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Nucleic Acids Res. 1997 Sep 1;25(17):3465-70. doi: 10.1093/nar/25.17.3465.
Obtaining pure mRNA preparations from prokaryotes has been difficult, if not impossible, for want of a poly(A) tail on these messages. We have used poly(A) polymerase from yeast to effect specific polyadenylation of Escherichia coli polysomal mRNA in the presence of magnesium and manganese. The polyadenylated total mRNA, which could be subsequently purified by binding to and elution from oligo(dT) beads, had a size range of 0.4-4.0 kb. We have used hybridization to a specific plasmid-encoded gene to further confirm that the polyadenylated species represented mRNA. Withdrawal of Mg2+ from the polyadenylation reaction resulted in addition of poly(A) to 16S rRNA despite the presence of Mn2+, indicating the vital role of Mg2+ in maintaining the native structure of polysomes. Complete dissociation of polysomes into ribosomal subunits resulted in quantitative polyadenylation of both 16S and 23S rRNA species. Chromosomal lacZ gene-derived messages were quantitatively recovered in the oligo(dT)-bound fraction, as demonstrated by RT-PCR analysis. Potential advantages that accrue from the availability of pure total mRNA from prokaryotes is discussed.
由于原核生物的信使RNA缺乏聚腺苷酸尾巴，要获得其纯mRNA制剂即便不是不可能，也一直很困难。我们利用酵母的聚腺苷酸聚合酶，在镁离子和锰离子存在的情况下，对大肠杆菌多核糖体mRNA进行特异性聚腺苷酸化。聚腺苷酸化的总mRNA随后可通过与寡聚（dT）磁珠结合并洗脱来纯化，其大小范围为0.4 - 4.0 kb。我们利用与特定质粒编码基因的杂交，进一步证实聚腺苷酸化的物种代表mRNA。从聚腺苷酸化反应中去除镁离子，尽管存在锰离子，仍导致16S核糖体RNA添加了聚腺苷酸，这表明镁离子在维持多核糖体天然结构中起着至关重要的作用。多核糖体完全解离成核糖体亚基导致16S和23S核糖体RNA物种都发生定量聚腺苷酸化。通过逆转录聚合酶链反应（RT-PCR）分析表明，染色体lacZ基因衍生的信使RNA在寡聚（dT）结合部分中被定量回收。文中讨论了从原核生物获得纯总mRNA所带来的潜在优势。
