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首页> 外文期刊>Molecular and Cellular Biology >The conserved U.G pair in the 5' splice site duplex of a group I intron is required in the first but not the second step of self-splicing.
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The conserved U.G pair in the 5' splice site duplex of a group I intron is required in the first but not the second step of self-splicing.

机译：I内含子的5'剪接位点双链体中的保守U.G对在自我剪接的第一步而非第二步中是必需的。

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Group I self-splicing introns have a 5' splice site duplex (P1) that contains a single conserved base pair (U.G). The U is the last nucleotide of the 5' exon, and the G is part of the internal guide sequence within the intron. Using site-specific mutagenesis and analysis of the rate and accuracy of splicing of the Tetrahymena thermophila group I intron, we found that both the U and the G of the U.G pair are important for the first step of self-splicing (attack of GTP at the 5' splice site). Mutation of the U to a purine activated cryptic 5' splice sites in which a U.G pair was restored; this result emphasizes the preference for a U.G at the splice site. Nevertheless, some splicing persisted at the normal site after introduction of a purine, suggesting that position within the P1 helix is another determinant of 5' splice site choice. When the U was changed to a C, the accuracy of splicing was not affected, but the Km for GTP was increased by a factor of 15 and the catalytic rate constant was decreased by a factor of 7. Substitution of U.A, U.U, G.G, or A.G for the conserved U.G decreased the rate of splicing by an even greater amount. In contrast, mutation of the conserved G enhanced the second step of splicing, as evidenced by a trans-splicing assay. Furthermore, a free 5' exon ending in A or C instead of the conserved U underwent efficient ligation. Thus, unlike the remainder of the P1 helix, which functions in both the first and second steps of self-splicing, the conserved U.G appears to be important only for the first step.
机译：I组自剪接内含子有一个5'剪接位点双链体（P1），其中包含一个保守的碱基对（U.G）。 U是5'外显子的最后一个核苷酸，而G是内含子内部指导序列的一部分。使用定点诱变并分析嗜热四膜膜虫第I内含子剪接的速率和准确性，我们发现UG对的U和G对自我剪接的第一步（在5'拼接位置）。将U突变为嘌呤激活的5'隐性剪接位点，并在其中还原U.G对;该结果强调了在拼接位点优先选择UG。然而，在引入嘌呤后，某些剪接仍保留在正常位点，这表明P1螺旋内的位置是5'剪接位点选择的另一个决定因素。当U更改为C时，剪接的准确性不会受到影响，但是GTP的Km增加了15倍，催化速率常数降低了7倍。UA，UU，GG，对于保守的UG，AG或AG的剪接率甚至降低了更多。相反，保守的G的突变增强了剪接的第二步，如反式剪接法所证明的。此外，以A或C而不是保守的U结尾的游离5'外显子进行有效的连接。因此，不同于P1螺旋的其余部分在自拼接的第一步和第二步均起作用，保守的U.G似乎仅对第一步很重要。

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