In vitro transcription and translational efficiency of chimeric SP6 messenger RNAs devoid of 5' vector nucleotides.
In vitro transcription and translational efficiency of chimeric SP6 messenger RNAs devoid of 5' vector nucleotides.
复制标题
缺乏 5 载体核苷酸的嵌合 SP6 信使 RNA 的体外转录和翻译效率。
DOI：
10.1093/nar/16.10.4483
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发表时间：
1988
影响因子：
14.9
通讯作者：
Gehrke,L
中科院分区：
文献类型：
--
作者：
Jobling,SA;Cuthbert,CM;Rogers,SG;Fraley,RT;Gehrke,L
一种含有噬菌体SP 6启动子的质粒，命名为pHSTO，用于无载体衍生核苷酸的RNA的体外转录。该载体已被表征为相对转录活性使用的结构，改变保守的核苷酸延伸超过SP 6转录起始位点。SP 6聚合酶有效地转录在相对于SP 6启动子的+1位含有鸟苷（G）核苷酸的cDNA插入物;然而，在+1位具有腺苷（A）或嘧啶的插入物不被转录。利用该载体，几种细胞和病毒的cDNA已被转录成可翻译的信使RNA;然而，SP 6聚合酶不能有效地转录苜蓿花叶病毒RNA 4的富含A-T的非翻译前导序列，除非病毒mRNA帽位点与转录起始位点被载体DNA的12个碱基对分开。通过将几种病毒mRNA的非翻译前导序列连接到大麦α-淀粉酶的编码区来产生嵌合信使RNA，并将所得mRNA在小麦胚芽提取物中翻译以确定相对翻译效率。芜菁黄花叶病毒外壳蛋白mRNA和黑甲虫病毒RNA 2的非翻译前导序列没有增加翻译效率，而烟草花叶病毒前导序列显著促进翻译。结果表明，同源非翻译前导序列被源自高效mRNA的前导序列取代不一定预示嵌合mRNA的翻译效率增强。
A plasmid containing the bacteriophage SP6 promoter, designated pHSTO, permitsin vitrotranscription of RNAs devoid of vector-derived nucleotides. This vector has been characterized for relative transcriptional activity using constructs which alter the conserved nucleotides extending beyond the SP6 transcriptional initiation site. SP6 polymerase efficiently transcribes cDNA inserts which contain a guanosine (G) nucleotide at position +1 relative to the SP6 promoter; however, inserts with an adenosine (A) or pyrimidine at position +1 are not transcribed. Several cellular and viral cDNAs have been transcribed into translatable messenger RNA using this vector; however, SP6 polymerase will not transcribe the A-T rich untranslated leader from alfalfa mosaic virus RNA 4 efficiently unless the viral mRNA cap site is separated from the transcriptional initiation site by twelve base pairs of vector DNA. Chimeric messenger RNAs were created by linking the untranslated leader sequence of several viral mRNAs to the coding region of barley α-amylase, and the resultant mRNAs were translated in a wheat germ extract to determine relative translational efficiencies. The untranslated leader sequences of turnip yellow mosaic virus coat protein mRNA and black beetle virus RNA 2 did not increase translational efficiency, while the tobacco mosaic virus leader stimulated translation significantly. The results indicate that substitution of a cognate untranslated leader sequence with a leader derived from a highly efficient mRNA does not necessarily predict enhanced translational efficiency of the chimeric mRNA.