Payvar F, Schimke R T
J Biol Chem. 1979 Aug 25;254(16):7636-42.
Translation of total mRNA in heterologous protein-synthesizing systems is often employed as an indirect means of assessing relative mRNA concentrations. However, it is well known that the efficiency of translation of specific mRNAs differs. One such example is the poor translational efficiency of conalbumin mRNA relative to ovalbumin mRNA. In this report we have studied the translation of conalbumin and ovalbumin mRNAs in crude mRNA preparations and with highly purified mRNA preparations. We find that treatment of RNA with methylmercury hydroxide prior to translation improves the translational efficiency of both mRNAs and preferentially improves translational efficiency of conalbumin mRNA to the point where it more correctly reflects the relative concentration of these two mRNAs in crude mRNA preparations. Conalbumin mRNA is also a poor template for the synthesis of full length cDNA synthesis by avian myeloblastosis virus reverse transcriptase, and treatment of this mRNA with methylmercury hydroxide increases the size of DNA sequences synthesized. We conclude that treatment with methylmercury hydroxide produces a partial denaturation of mRNA complexed with either itself or with other RNA molecules and results in more efficient utilization in both translational assays and DNA polymerization reactions.
在异源蛋白质合成系统中,总mRNA的翻译常被用作评估相对mRNA浓度的间接手段。然而,众所周知,特定mRNA的翻译效率有所不同。一个这样的例子是伴清蛋白mRNA相对于卵清蛋白mRNA的翻译效率较低。在本报告中,我们研究了伴清蛋白和卵清蛋白mRNA在粗制mRNA制剂以及高度纯化的mRNA制剂中的翻译情况。我们发现,在翻译前用氢氧化甲基汞处理RNA可提高这两种mRNA的翻译效率,并且优先提高伴清蛋白mRNA的翻译效率,使其更准确地反映粗制mRNA制剂中这两种mRNA的相对浓度。伴清蛋白mRNA也是禽成髓细胞瘤病毒逆转录酶合成全长cDNA的不良模板,用氢氧化甲基汞处理该mRNA可增加合成的DNA序列长度。我们得出结论,用氢氧化甲基汞处理会使与自身或其他RNA分子复合的mRNA发生部分变性,并导致在翻译测定和DNA聚合反应中都能更有效地利用。