Francis M A, Rajbhandary U L
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Mol Cell Biol. 1990 Sep;10(9):4486-94. doi: 10.1128/mcb.10.9.4486-4494.1990.
We showed previously that the human initiator tRNA gene, in the context of its own 5'- and 3'-flanking sequences, was not expressed in Saccharomyces cerevisiae. Here we show that switching its 5'-flanking sequence with that of a yeast arginine tRNA gene allows its functional expression in yeast cells. The human initiator tRNA coding sequence was either cloned downstream of the yeast arginine tRNA gene, with various lengths of intergenic spacer separating them, or linked directly to the 5'-flanking sequence of the yeast arginine tRNA coding sequence. The human initiator tRNA made in yeast cells can be aminoacylated with methionine, and it was clearly separated from the yeast initiator and elongator methionine tRNAs by RPC-5 column chromatography. It was also functional in yeast cells. Expression of the human initiator tRNA in transformants of a slow-growing mutant yeast strain, in which three of the four endogenous initiator tRNA genes had been inactivated by gene disruption, resulted in enhancement of the growth rate. The degree of growth rate enhancement correlated with the steady-state levels of human tRNA in the transformants. Besides providing a possible assay for in vivo function of mutant human initiator tRNAs, this work represents the only example of the functional expression of a vertebrate RNA polymerase III-transcribed gene in yeast cells.
我们之前表明，人类起始tRNA基因在其自身的5'和3'侧翼序列的背景下，在酿酒酵母中不表达。在此我们表明，将其5'侧翼序列与酵母精氨酸tRNA基因的5'侧翼序列进行交换，可使其在酵母细胞中实现功能性表达。人类起始tRNA编码序列要么克隆在酵母精氨酸tRNA基因的下游，二者之间有不同长度的基因间隔区隔开，要么直接与酵母精氨酸tRNA编码序列的5'侧翼序列相连。在酵母细胞中产生的人类起始tRNA能够用甲硫氨酸进行氨酰化，并且通过RPC - 5柱色谱法可明显地将其与酵母起始和延伸甲硫氨酸tRNA区分开来。它在酵母细胞中也具有功能。在一种生长缓慢的突变酵母菌株的转化体中表达人类起始tRNA，该菌株的四个内源性起始tRNA基因中有三个已通过基因破坏而失活，结果导致生长速率提高。生长速率提高的程度与转化体中人类tRNA的稳态水平相关。除了为突变型人类起始tRNA的体内功能提供一种可能的检测方法外，这项工作代表了脊椎动物RNA聚合酶III转录基因在酵母细胞中功能性表达的唯一实例。
