An extended RNA binding site for the yeast branch point-binding protein and the role of its zinc knuckle domains in RNA binding
An extended RNA binding site for the yeast branch point-binding protein and the role of its zinc knuckle domains in RNA binding
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DOI：
10.1074/jbc.m603137200
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发表时间：
2006-09-15
影响因子：
4.8
通讯作者：
Berglund, J. Andrew
中科院分区：
文献类型：
--
作者：
Garrey, Stephen M.;Voelker, Rodger;Berglund, J. Andrew
酿酒酵母中UAC-UAAC的高度保守的分支点序列(BPS)最初被分支点结合蛋白(BBP)识别。通过指数富集法对配体的系统进化，我们已经确定酵母BBP与亲和力最高的分支点序列UACUAAC结合，并偏好BPS下游的额外腺苷。此外，我们还发现，BPS上游的茎环增强了与人工设计的RNA的结合(30倍效应)和与酵母内含子RNA的结合(3倍效应)。BBP的锌指关节部分负责增强与茎环的结合，但似乎在BBP与单链RNA底物的结合中没有明显作用。BBP的C-末端缺失表明，两个锌指关节之间以及N-末端RNA结合域(KH和QUA2结构域)与第一个锌指关节之间的连接区对于与RNA结合是重要的。第二个高度保守的锌指关节在RNA结合中没有参与，这表明该锌指关节在RNA加工中起着不同于增强BBP与BPS结合的作用。
The highly conserved branch point sequence (BPS) of UAC-UAAC in Saccharomyces cerevisiae is initially recognized by the branch point-binding protein (BBP). Using systematic evolution of ligands by exponential enrichment we have determined that yeast BBP binds the branch point sequence UACUAAC with highest affinity and prefers an additional adenosine downstream of the BPS. Furthermore, we also found that a stem-loop upstream of the BPS enhances binding both to an artificially designed RNA(30-fold effect) and to an RNA from a yeast intron (3-fold effect). The zinc knuckles of BBP are partially responsible for the enhanced binding to the stem-loop but do not appear to have a significant role in the binding of BBP to single-strand RNA substrates. C-terminal deletions of BBP reveal that the linker regions between the two zinc knuckles and between the N-terminal RNA binding domains (KH and QUA2 domains) and the first zinc knuckle are important for binding to RNA. The lack of involvement of the second highly conserved zinc knuckle in RNA binding suggests that this zinc knuckle plays a different role in RNA processing than enhancing the binding of BBP to the BPS.