Sprague K U, Larson D, Morton D
Cell. 1980 Nov;22(1 Pt 1):171-8. doi: 10.1016/0092-8674(80)90165-8.
We have used homologous in vitro transcription to examine the nucleotide sequences required for activity of cloned Bombyx mori tRNA2Ala genes. We have compared the transcriptional properties of an intact gene and a truncated derivative of the same gene lacking all but 11 nucleotides of normal 5' flanking DNA, and we find that at least two regions of DNA are required for accurate transcription of tRNA2Ala genes by homologous B. mori extracts. One of these sites is retained in the truncated gene and may be within the tRNA coding sequence. Competition experiments with intact and truncated genes indicate that this site probably acts by binding a factor necessary for specificity in polymerase III-catalyzed transcription. The second required site is removed by the Hind III cleavage used to produce shortened genes and thus must be 11 nucleotides or more upstream from the transcription initiation point. Possible roles for this site are discussed.
我们利用同源体外转录来研究克隆的家蚕tRNA2Ala基因活性所需的核苷酸序列。我们比较了一个完整基因和同一基因的截短衍生物的转录特性，该截短衍生物除了正常5'侧翼DNA的11个核苷酸外其余全部缺失，我们发现家蚕同源提取物准确转录tRNA2Ala基因至少需要两个DNA区域。其中一个位点保留在截短基因中，可能位于tRNA编码序列内。完整基因和截短基因的竞争实验表明，该位点可能通过结合聚合酶III催化转录特异性所需的因子发挥作用。产生缩短基因所用的Hind III切割去除了第二个必需位点，因此该位点必须位于转录起始点上游11个核苷酸或更多。讨论了该位点可能的作用。
