Greer C L, Söll D, Willis I
Mol Cell Biol. 1987 Jan;7(1):76-84. doi: 10.1128/mcb.7.1.76-84.1987.
We have examined the substrate requirements for efficient and accurate splicing of tRNA precursors in Saccharomyces cerevisiae. The effects of Schizosaccharomyces pombe tRNASer gene mutations on the two steps in splicing, intron excision and joining of tRNA halves, were determined independently by using partially purified splicing endonuclease and tRNA ligase from S. cerevisiae. Two mutations (G14 and A46) reduced the efficiency of excision and joining in parallel, whereas two others (U47:7 and C33) produced differential effects on these two steps; U47:7 affected primarily the excision reaction, and C33 had a greater impact on ligation. These data indicate that endonuclease and ligase recognize both common and unique features of their substrates. Another two mutations (Ai26 and A37:13) induced miscutting, although with converse effects on the two splice sites. Thus, the two cutting events appear to be independent. Finally, we suggest that splice sites may be determined largely through their position relative to sites within the tRNA-like domain of the precursors. Several of these important sites were identified, and others are proposed based on the data described here.
我们研究了酿酒酵母中tRNA前体高效准确剪接的底物需求。通过使用来自酿酒酵母的部分纯化的剪接内切核酸酶和tRNA连接酶，分别确定了粟酒裂殖酵母tRNASer基因突变对剪接的两个步骤（内含子切除和tRNA两半的连接）的影响。两个突变（G14和A46）平行降低了切除和连接的效率，而另外两个突变（U47:7和C33）对这两个步骤产生了不同的影响；U47:7主要影响切除反应，而C33对连接有更大的影响。这些数据表明内切核酸酶和连接酶识别其底物的共同特征和独特特征。另外两个突变（Ai26和A37:13）诱导了错误切割，尽管对两个剪接位点有相反的影响。因此，这两个切割事件似乎是独立的。最后，我们认为剪接位点可能很大程度上是由它们相对于前体tRNA样结构域内位点的位置决定的。确定了其中几个重要位点，并根据此处描述的数据提出了其他位点。
