FUNCTIONAL-CHARACTERIZATION OF AN INOSITOL-SENSITIVE UPSTREAM ACTIVATION SEQUENCE IN YEAST - A CIS-REGULATORY ELEMENT RESPONSIBLE FOR INOSITOL-CHOLINE MEDIATED REGULATION OF PHOSPHOLIPID BIOSYNTHESIS
FUNCTIONAL-CHARACTERIZATION OF AN INOSITOL-SENSITIVE UPSTREAM ACTIVATION SEQUENCE IN YEAST - A CIS-REGULATORY ELEMENT RESPONSIBLE FOR INOSITOL-CHOLINE MEDIATED REGULATION OF PHOSPHOLIPID BIOSYNTHESIS
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DOI：
10.1074/jbc.270.42.25087
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发表时间：
1995-10-20
影响因子：
4.8
通讯作者：
HENRY, SA
中科院分区：
文献类型：
--
作者：
BACHHAWAT, N;OUYANG, QA;HENRY, SA
在编码磷脂生物合成酶的基因启动子中存在一个重复元件，肌醇敏感上游激活序列(UAS(INO))，具有共同的序列5‘-CATGTGAAAT-3’，该基因受磷脂前体肌醇和胆碱的调节。没有一个自然产生的UAS(INO)元素的变体准确地概括了共识(有关审查，请参见Carman，G.M.和Henry，S.A.(1989)Annu。Biochem牧师。58,635-669和Paltauf，F.，Kolwhin，S.和Henry，S.A.(1992)在酵母分子生物学中(Broach，J.，Jones，E.和Pringle，J.，编辑)第2卷，冷泉港实验室，冷泉港，NY)。UAS(INO)元件的前六个碱基与碱性螺旋-环螺旋(BHLH)家族蛋白质的典型结合基序同源。酵母中的两种bHLH调节蛋白Ino2p和Ino4p与含有该元件的启动子片段结合。本研究对UAS(INO)的功能进行了广泛的分析。我们报道，在体内表达试验中，假定的bHLH结合位点内的任何碱基替换都会导致UAS(INO)功能的急剧减少或完全消失。还评估了10碱基对重复序列两侧5‘区域的碱基替换，以及重复序列本身在bHLH核心外的3’端的序列。事实证明，紧接在元件5‘端两侧的两个碱基对其作为UAS元件的功能非常重要，bHLH核心基序的两个碱基立即3’端也是如此。替换原来10个碱基对共识的最后两个碱基(即5‘-CATGTGAAAT-3’)没有那么戏剧性的影响。我们还测试了改变的元件的子集在Ino2p分析中作为竞争对手的能力。Ino4p结合。体内测定的任何给定序列作为UAS(INO)元件的强度与其作为Ino2p竞争者的强度密切相关。Ino4p结合。我们还测试了修改的UAS(INO)元件的子集对携带阿片1突变的菌株体内表达的影响。在磷脂前体存在的情况下，阿片1突变使磷脂合成的共调控酶具有结构性。在野生型菌株中测试时，所有保留了一些残留UAS(INO)活性的元素都在成分上表达，其表达水平与在opi1突变体中测试时野生型去抑制水平相当。因此，UAS(INO)似乎负责OPI1介导的抑制，以及Ino2p。Ino4p结合。此外，UAS(INO)元件的每个已识别功能似乎都具有相同的序列特异性，并且都需要完整的bHLH基序的存在，这表明转录激活、抑制和Ino2p。Ino4p结合都是单一调控机制的组成部分。
A repeated element, the inositol-sensitive upstream activation sequence (UAS(INO)), having the consensus sequence, 5'-CATGTGAAAT-3', is present in the promoters of genes encoding enzymes of phospholipid biosynthesis that are regulated in response to the phospholipid precursors, inositol and choline. None of the naturally occurring variants of the UAS(INO) element exactly recapitulates the consensus (for review, see Carman, G. M., and Henry, S. A. (1989) Annu. Rev. Biochem. 58, 635-669 and Paltauf, F., Kolwhein, S., and Henry, S. A. (1992) in Molecular Biology of the Yeast Saccharomyces cerevisiae (Broach, J., Jones, E., and Pringle, J., eds) Vol. 2, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The first six bases of the UAS(INO) element are homologous with canonical binding motif for proteins of the basic helix-loop helix (bHLH) family. Two bHLH regulatory proteins, Ino2p and Ino4p from yeast, were previously shown to bind to promoter fragments containing this element.In the present study, an extensive analysis of UAS(INO) function has been conducted. We report that any base substitution within the putative bHLH binding site resulted either in a dramatic reduction or in a complete obliteration of UAS(INO) function as tested in an expression assay in vivo. Base substitutions in the 5' region that flanks the 10-base pair repeat, as well as sequences within the repeat itself at its 3' end outside the bHLH core, were also assessed. The two bases immediately flanking the 5' end of the element proved to be very important to its function as a UAS element as did the two bases immediately 3' of the bHLH core motif. Substitutions of the final two bases of the original ten base pair consensus (i.e. 5'-CATGTGAAAT-3') had less dramatic effects.We also tested a subset of the altered elements for their ability to serve as competitors in an assay of Ino2p . Ino4p binding. The strength of any given sequence as a UAS(INO) element, as assayed in vivo, was strongly correlated with its strength as a competitor for Ino2p . Ino4p binding. We also tested a subset of the modified UAS(INO) elements for their effects on expression in vivo in a strain carrying an opi1 mutation. The opi1 mutation renders the coregulated enzymes of phospholipid synthesis constitutive in the presence of phospholipid precursors. All elements that retained some residual UAS(INO) activity when tested in the wild-type strain were constitutively expressed at a level comparable with the wild-type derepressed level when tested in the opi1 mutant. Thus, UAS(INO) appears to be responsible for OPI1 mediated repression, as well as Ino2p . Ino4p binding. Furthermore, each of the identified functions of the UAS(INO) element appears to have the same sequence specificity, and all require the presence of the intact bHLH motif, suggesting that transcriptional activation, repression, and Ino2p . Ino4p binding are all components of a single regulatory mechanism.