Cao J, Geballe A P
Division of Molecular Medicine, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
RNA. 1998 Feb;4(2):181-8.
A 22-codon upstream open reading frame (uORF2) in the human cytomegalovirus UL4 transcript leader inhibits downstream translation in cis. Previous studies revealed that the peptide product of uORF2 mediates this inhibitory effect by interfering with translation termination at its own stop codon. The block at termination results both in accumulation of the nascent uORF2 peptide linked to tRNA(Pro), the tRNA that decodes the final codon of uORF2, and in stalling of ribosomes at the end of uORF2. The stalled ribosomes create a barrier that obstructs ribosomal transit to the downstream cistron. In the current studies, we further investigated the mechanism of uORF2-mediated translational inhibition by assessing the kinetics of uORF2 peptidyl tRNA(Pro) hydrolysis and ribosomal release from the uORF2 termination site. Whereas hydrolysis of a mutant, noninhibitory uORF2 peptidyl tRNA is nearly complete in less than 1 min, hydrolysis of the wild-type peptidyl tRNA is negligible even after 30 min. In spite of this remarkably prolonged block to hydrolysis of the uORF2 peptidyl tRNA(Pro), most ribosomes are released from the uORF2 termination site within 15 min. Thus, peptidyl tRNA hydrolysis is not absolutely required for ribosomal release in this system. These results suggest that a eukaryotic cellular mechanism exists for removing stalled ribosomes from mRNAs in the absence of peptidyl tRNA hydrolysis.
人巨细胞病毒UL4转录本前导区中一个22密码子的上游开放阅读框（uORF2）以顺式作用抑制下游翻译。先前的研究表明，uORF2的肽产物通过干扰其自身终止密码子处的翻译终止来介导这种抑制作用。终止受阻导致与tRNA(Pro)相连的新生uORF2肽积累，tRNA(Pro)是解码uORF2最后一个密码子的tRNA，同时也导致核糖体在uORF2末端停滞。停滞的核糖体形成了一个屏障，阻碍核糖体向下游顺反子移动。在当前的研究中，我们通过评估uORF2肽基tRNA(Pro)水解动力学以及核糖体从uORF2终止位点释放的动力学，进一步研究了uORF2介导的翻译抑制机制。虽然突变的、无抑制作用的uORF2肽基tRNA在不到1分钟内几乎完全水解，但即使在30分钟后，野生型肽基tRNA的水解也可以忽略不计。尽管uORF2肽基tRNA(Pro)的水解受到显著延长的阻滞，但大多数核糖体在15分钟内从uORF2终止位点释放。因此，在这个系统中，核糖体释放并不绝对需要肽基tRNA水解。这些结果表明，存在一种真核细胞机制，可在没有肽基tRNA水解的情况下从mRNA中去除停滞的核糖体。
