Conformational switches involved in orchestrating the successive steps of group I RNA splicing
Conformational switches involved in orchestrating the successive steps of group I RNA splicing
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DOI：
10.1021/bi952599z
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发表时间：
1996-03-26
期刊：
影响因子：
2.9
通讯作者：
Cech, TR
中科院分区：
文献类型：
--
作者：
Golden, BL;Cech, TR
第一组内含子在其3‘端有一个保守的鸟苷残基，称为omega G，对于四膜虫前-rRNA来说，这是第二步剪接的主要决定因素。我们研究了omega G在鱼腥藻PCC7120 tRNA(Leu)前体的249个残基I内含子自剪接中的作用。与四膜虫Pre-rRNA内含子的观察相反，在omega位置放置腺苷残基的突变对剪接的第二步没有严重影响；3‘剪接位点的选择和第二步的速率都没有改变。然而，拼接的第一步现在很容易被逆转。这种意想不到的效果也是因为突变改变了内含子鸟苷结合位点的核苷专一性。这些突变的共同主题是，当鸟苷结合位点和omega残基之间没有强烈的相互作用时，剪接的第一步会发生逆转。这表明omega G的主要作用是在剪接的第一步与添加到内含子中的外源鸟苷分子竞争内含子的单个鸟苷结合部位，根据这些数据，我们能够通过在剪接的第一步和第二步之间提出两个不同的构象变化来扩展该内含子的自剪接反应的机制。第一个是外源核苷与omega核苷的交换。这就是我们可以被omega位置或鸟苷结合位点的突变扰乱的平衡，额外的构象变化然后完全激活第二步剪接的内含子。
Group I introns possess a conserved guanosine residue at their 3' end, termed omega G, that, in the case of the Tetramhymena pre-rRNA, is a major determinant of the second step of splicing, We examined the role of omega G in self-splicing of the 249-residue group I intron of the Anabaena PCC7120 tRNA(leu) precursor. Contrary to observations with the Tetrahymena pre-rRNA intron, a mutation that places an adenosine residue at the omega position did not have a severe effect on the second step of splicing; neither 3' splice-site selection nor the rate of the second step was altered. The first step of splicing, however, was now readily reversed. This unexpected effect also resulted from a mutation that altered the nucleoside specificity of the intronic guanosine-binding site. The theme common to these mutations is that reversal of the first step of splicing results when there is not a strong interaction between the guanosine-binding site and the omega residue. This suggests that a major role of omega G is to compete with the exogenous guanosine molecule added to the intron in the first step of splicing for the single guanosine-binding site of the intron, From these data, we are able to extend the mechanism for the self-splicing reaction of this intron by proposing two distinct conformational changes between the first and second steps of splicing. The first of these is the exchange of the exogenous nucleoside for the omega nucleoside. This is the equilibrium that we can perturb by mutations at either the omega position or the guanosine-binding site, An additional conformational change then fully activates the intron for the second step of splicing.