TERMINATION-REINITIATION OCCURS IN THE TRANSLATION OF MAMMALIAN-CELL MESSENGER-RNAS
TERMINATION-REINITIATION OCCURS IN THE TRANSLATION OF MAMMALIAN-CELL MESSENGER-RNAS
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DOI：
10.1128/mcb.6.7.2695
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发表时间：
1986-07-01
影响因子：
5.3
通讯作者：
BERG, P
中科院分区：
文献类型：
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作者：
PEABODY, DS;BERG, P
近年来，真核生物内部翻译启动的例子层出不穷。在许多情况下，上游阅读框的终止子在内部起始点之前，这表明翻译重新启动可能是AUGS间隔启动的一种机制。为了验证这一想法，我们在哺乳动物表达载体pSV2中构建了一系列重组体。每一个都含有一个双顺反子转录单位，该单位包括小鼠二氢叶酸还原酶(DHFR)的编码序列和来自大肠杆菌的黄嘌呤-鸟嘌呤磷酸核糖基转移酶(XGPRT)基因。不同版本的pSV2dhfr-gpt重组质粒改变了DHFR阅读框相对于XGPRT起始密码子的终止位置，表明这是XGPRT活性在转基因Cos-1细胞中表达的关键因素。因此，当DHFR帧终止于XGPRT启动器AUG的上游或下游非常短的距离时，观察到相当水平的XGPRT活性。当DHFR框架终止超过XGPRT启动子的50个核苷酸时，活性降低了大约两倍。然而，当DHFR和XGPRT序列在框内融合，使得在DHFR AUG开始的核糖体直到它们遇到XGPRT终止子才终止时，XGPRT活性的产生被取消。这种内部翻译起始对上游阅读框架终止子位置的依赖与哺乳动物核糖体能够翻译重新起始的假设是一致的。
Many examples of internal translation initiation in eucaryotes have accumulated in recent years. In many cases terminators of upstream reading frames precede the internal initiation site, suggesting that translational reinitiation may be a mechanism for initiation at interval AUGs. To test this idea, a series of recombinants was constructed in the mammalian expression vector pSV2. Each contained a dicistronic transcription unit comprising the coding sequence for mouse dihydrofolate reductase (DHFR) followed by the gene for xanthine-guanine phosphoribosyl transferase (XGPRT) from Escherichia coli. Various versions of this pSV2dhfr-gpt recombinant plasmid altered the location at which the DHFR reading frame was terminated relative to the XGPRT initiation codon and demonstrated that this is a critical factor for the expression of XGPRT activity in transfected Cos-1 cells. Thus, when the DHFR frame terminated upstream or a very short distance downstream of the XGPRT initiator AUG, substantial levels of XGPRT activity were observed. When the DHFR frame terminated 50 nucleotides beyond the XGPRT initiator, activity was reduced about twofold. However, when the DHFR and XGPRT sequences were fused in-frame so that ribosomes which initiated at the DHFR AUG did not terminate until they encountered the XGPRT terminator, production of XGPRT activity was abolished. This dependence of internal translation initiation on the position of terminators of the upstream reading frame is consistent with the hypothesis that mammalian ribosomes are capable of translational reinitiation.