酵母半乳糖代谢基因多重转录调控机制的研究
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摘要
酵母半乳糖代谢基因受半乳糖诱导表达,并且依据外界碳源的不同有三种基因表达方式：当以葡萄糖作为碳源时,与葡萄糖有关的各种抑制元件如Mig1可以结合在GAL基因上游的抑制元件结合区域抑制GAL基因的表达；另一方面,转录激活因子Gal4虽然能够结合在UAS序列上,但此时转录抑制因子Gal80结合在其转录激活区域(TAD),抑制着转录激活因子Gal4的活性,从而抑制GAL基因的转录,这时GAL基因处于抑制状态；甘油／乳酸盐或棉子糖等非发酵碳源培养时,即细胞处在非抑制非诱导条件时,各种葡萄糖抑制元件从GAL基因上游离开,解除了葡萄糖抑制效应,但此时Gal80仍然结合在Gal4的转录激活区域,抑制着Gal4的转录活性,这时GAL结构基因仍然不能大量表达。然而与抑制条件不同,此时半乳糖信号感应蛋白Gal3已经有表达了,所以此时GAL只有基础水平的转录；半乳糖做为碳源时,半乳糖,ATP以及信号感应蛋白Gal3能够与转录抑制因子Gal80结合,使得Gal80能够从结合在酵母半乳糖代谢基因gal的上游激活序列(UAS)上的GAL4／GAL80复合物上脱落,使得转录激活因子Gal4激活区域得以暴露,从而可以募集RNA聚合酶以及各种与转录有关的辅助因子,进而启动下游的参与半乳糖分解代谢有关酶的基因的表达。这种诱导作用体现在快速和高效上,几分钟就有高达1000倍以上的转录水平上的提升；在非抑制非诱导和诱导条件下,转录激活因子Gal4激活gal基因的转录的过程中伴随着Gal4本身的降解.这种降解作用是依赖于泛素介导的蛋白酶体途径实现的,在整个途径中,泛素连接酶起到对靶蛋白特异性识别的作用：在非诱导条件下,有转录激活作用的Gal4的837位丝氨酸被与RNA聚合酶Ⅱ全酶有关的激酶Kin28磷酸化,泛素连接酶(E3)Grr1识别这种磷酸化修饰的Gal4,在泛素激活酶(E1),泛素结合酶(E2)的作用下实现Gal4的多泛素化,给其打上被降解的标签,通过26S蛋白酶体来降解。经研究表明,在△Grr1菌株中,Gal4不能被正常的降解而有着一定程度的积累,此时GAL基因的表达水平有着几十倍的提升,说明了Gal4的积累造成了GAL基因表达水平的升高,这时泛素连接酶Grr1介导的Gal4的降解方式与Gal4的转录活性的调控无关；在诱导条件下,有转录激活活性的Gal4的699和837位的丝氨酸分别被与RNA聚合酶Ⅱ全酶有关的激酶Srb10和Kin28磷酸化,泛素连接酶Dsg1识别有活性的磷酸化的Gal4,使其多泛素化进而被蛋白酶体降解。然而在△dsg1菌株中,磷酸化修饰的Gal4虽然能够大量积累,GAL基因也能够大量的转录产生mRNA,但生成的mRNA都是有缺陷的并不能正常的进行翻译,所以这是菌体表现为不能够利用半乳糖。可见与Grr1介导的Gal4的降解方式不同,Dsg1介导的Gal4的降解方式还与基因的表达调控有关,很可能泛素连接酶Dsg1本身出了识别降解Gal4外,本身还直接参与了GAL基因的转录调控过程。此外通过对转录激活因子Gal4的翻译后修饰-单泛素化可以稳定19S蛋白酶体亚基在启动子上的结合,从而调控GAL基因正常表达；单泛素化修饰的缺陷直接导致GAL基因的诱导表达缺陷。可见泛素-蛋白酶体这条降解途径除了降解靶蛋白的同时还发挥了非降解性的调控作用：对酵母半乳糖代谢基因的转录从不同阶段不同步骤中发挥了重要的调控作用。这无疑是一种全新的调控机制,对于真核生物基因复杂的转录调控机制的探索有着重要的作用。
petite mutant亦简称小菌落,是酵母菌的一种突变型。在六七十年代,就发现了小菌落在半乳糖,麦芽糖等碳源上生长的缺陷,从而提出了线粒体中有控制着对不同糖利用的因子。而诱导条件下介导Gal4降解的Dsg1还控制着与线粒体外膜融合相关的Fzo1(GTPase)的降解,Dsg1的缺失同时也造成了线粒体形态的异常。因而Dsg1对于半乳糖代谢的影响是否与线粒体的功能异常有关还不清楚；线粒体中存在着催化物质代谢和能量转换的各种酶和辅酶,因而供能物质(如糖酵解产物丙酮酸)在线粒体内能得到彻底氧化分解,生成更多的高能磷酸化合物ATP以备细胞其它生命活动需要。细胞生命活动中所需能量约有95%来自线粒体。因此,线粒体的主要功能是进行细胞的氧化供能,故有细胞内“动力工厂”之称。在半乳糖代谢过程中,ATP直接参与了GAL基因的表达调控,这也从另一方面暗示了线粒体的产能与半乳糖的正常利用有关。另一方面,NADPH是生物体的一种重要辅酶,它不仅在生物合成反应中负责提供还原力,而且对于细胞抵抗活性氧的毒害也有重要作用。最近有研究表明,辅酶因子NAD(H)/NADP(H)能够直接和转录抑制因子Gal80结合来直接调控Gal4和Gal80之间的关系：当NAD(H)与Gal80结合时,能够稳定Gal4-Gal80之间的关系；而当NADP(H)与Gal80相结合时,能够去稳定Gal4-Gal80之间的关系从而有利于GAL基因的表达。而线粒体又是提供还原力的主要场所,能为细胞抗氧化做很大的贡献。因而线粒体中的NAD(H)/NADP(H)水平是否特异性调控GAL基因的表达成为了本论文研究的一个重点,对于更深层次揭示GAL基因调控机制有着重要的作用。作为单细胞真核生物的酿酒酵母,本身不仅具有高度的遗传可操作性,而且过去几十年以其作为模式生物的研究已经使人们对一些基础的细胞生物学过程及人类一些复杂疾病,如Creutzfel-Jacob病、Parkinson's病及癌症等发生机制的认识上取得了许多关键性的进展。与此同时,酿酒酵母作为分子生物学研究的三大模式生物之一,对许多基因的转录调控相关问题的研究具有其它生物无可比拟的独特优势。本论文工作在对半乳糖代谢调控进行较为系统研究的基础上,就转录激活因子Gal4的新的活性调控方式以及Gal4-Gal80相互作用关系对GAL基因的调控上进行了系统研究,对可能的作用机制进行了初步探讨。本文主要的工作内容及结果如下：
1.通过构建不同的基因缺失菌株和Gal4的定点突变或是部分缺失突变体,从遗传学角度和体外的实验探索了Gal4-Gal80之间的相互作用模式的改变对Dsg1调控GAL基因的影响机制
通过检测Gal4的699位丝氨酸,837位丝氨酸和两个位点都突变的点突变体在野生型菌株和△dsg1菌株中的报告基因的活性显示,这两个位点的磷酸化对于Dsg1的调控没有影响,即Dsg1对于GAL基因的调控并不是通过这两个位点的磷酸化来实现的；与以前文献报道不同,在半乳糖为碳源的平板的生长实验表明△dsg1菌株与野生型菌株相比,表现出了延迟生长的表型而不是不生长,这也与△dsg1菌株中的GAL基因诱导曲线相符,很可能Dsg1只是影响了GAL基因的初期诱导。Western实验结果表明,Dsg1的缺失并没有影响Gal4的泛素化,一方面Dsgl影响GAL基因表达不是通过对Gal4的泛素化来实现的,另一方面很可能存在有其他的泛素化Gal4的泛素连接酶。由于Dsg1还特异性识别降解与线粒体外膜融合相关的蛋白Fzo1,因而Dsg1缺失造成了线粒体形态的异常。但与线粒体分裂相关的基因dnm1缺失时能够部分回补线粒体形态上的不正常,通过检测△dnm1,△dnm1△dsg1菌株中的报告基因活性发现,线粒体形态的部分恢复能够部分恢复GAL基因的诱导表达,暗示了Dsg1对GAL基因的调控可能与线粒体的功能异常有关。当在gal80缺陷菌株或是不与Gal80相互作用或相互作用减弱的Gal4的突变体中,检测报告基因的活性发现△dsgl菌株中GAL基因恢复了正常的诱导；同时体内的酵母双杂交实验和体外的实验证明了回补Dsg1缺失的Gal4的突变体确实与转录抑制因子Gal80之间的相互作用减弱了。当检测与Gal80恢复正常的相互作用的Gal4的突变体的报告基因时,Dsg1缺失所造成的GAL基因诱导缺陷又存在了,充分说明了Gal4-Gal80之间的相互作用模式改变对Dsg1调控GAL基因有很大的影响。
2.通过构建线粒体bc1复合物的RIP1基因缺失菌株和ATP合成酶的ATP1基因缺失菌株,检测当酵母细胞胞内能量状态异常时对于GAL基因表达的影响及其机制
通过检测△rip1,△atp1菌株中的报告基因活性,结果显示GAL基因没有表达。提取△rip1,△atp1菌株中的mRNA,检测GAL1的(?)nRNA是否有转录,结果表明,△rip1,△atp1菌株中没有mRNA生成；CHIP实验进一步表明无论是RNA聚合酶还是羧基末端功能区5-丝氨酸磷酸化的RNA聚合酶都不能正常的结合启动子,造成GAL基因不能转录,从而△rip1菌株不能够正常的利用半乳糖。为了确定△rip1和△atp1基因是否真正影响了细胞内的ATP水平,我检测了基因缺失菌株中ATP浓度,结果显示胞内的ATP浓度与野生型菌株相比确实下降了很多；由此可以推断△rip1和△atp1由于影响了ATP的合成,造成了胞内ATP浓度的降低从而引起了GAL基因的转录缺陷。因为胞内各种代谢途径都会受到ATP水平的影响,因而RIP1和ATP1基因的缺失对半乳糖代谢的影响是否具有特异性?通过进一步对组成型表达基因ADH1和PMA1的mRNA的检测,结果显示ADH1和PAA1在野生型和△rip1和△atp1菌株均能正常的转录；葡萄糖和半乳糖为碳源的平板生长结果表明,野生型和△rip1和△atp1菌株均能正常的在以葡萄糖为碳源的平板生长,而以半乳糖为碳源的平板上△rip1和△atp1菌株均不能正常的生长。以上结果表明了RIP1和ATP1基因的缺失引起的胞内ATP水平的降低能够特异性的导致GAL基因转录缺陷。
3.通过构建线粒体中NAD(H)激酶POS5基因缺失菌株和细胞质中NAD激酶UTRl的基因缺失菌株,检测酵母细胞胞内NAD(H)／NADP(H)水平对GAL基因表达的影响及其机制
通过检测Δpos5和Δutr1菌株中的报告基因活性,结果显示GAL基因没有表达。提取Δpos5和Δutr1菌株中的mRNA,检测GAL1的mRNA是否有转录,结果表明,Δpos5和Δutr1菌株中没有mRNA生成；CHIP实验进一步表明无论是RNA聚合酶还是羧基末端功能区5-丝氨酸磷酸化的RNA聚合酶都不能正常的结合启动子,造成GAL基因不能转录,从而Δpos5菌株不能够正常的利用半乳糖。为了确定POS5和UTR1基因是否真正影响了细胞内NAD(H)／NADP(H)的水平,通过检测基因缺失菌株中NAD(H)／NADP(H)浓度,结果显示胞内的NAD(H)浓度与野生型菌株相比基本没有影响,而胞内NADP(H)的水平确实下降了很多,分别以葡萄糖和半乳糖为碳源的平板上对不同浓度H202的敏感性实验说明了Δpos5和Δutr1菌株与野生型相比抗氧化能力有了很大的降低,这很可能与胞内NADP(H)的水平下降有关；由此可以推断Δpos5和Δutr1基因缺失菌株由于影响了胞内NADP(H)浓度,造成了GAL基因的转录缺陷。因为胞内各种代谢途径都会受到NADP(H)水平的影响,因而POS5和UTR1基因的缺失对半乳糖代谢的影响是否具有特异性?通过进一步对组成型表达基因ADH1和PMA1的mRNA的检测,结果显示ADH1和PMA1在野生型和Δpos5和Δutr1菌株中均能正常的转录；葡萄糖和半乳糖为碳源的平板生长结果表明,野生型,Δpos5和Δutr1菌株均能正常的在以葡萄糖为碳源的平板生长,而以半乳糖为碳源的平板上Δpos5和Δutr1菌株均不能正常的生长。以上结果表明了POS5和UTR1基因的缺失引起的胞内NADP(H)水平的降低能够特异性的导致GAL基因转录缺陷。
4.在ATP合成途径基因和NAD(H)激酶基因缺失菌中过量表达Gal3或与Gal80相互作用改变的Gal4突变体与半乳糖代谢功能的恢复机制的研究
在半乳糖正常的代谢过程中,GAL基因的正常调控表达是必须的。GAL基因的诱导表达受转录激活因子Gal4和转录抑制因子Gal80,信号感应蛋白Gal3还有半乳糖,ATP小分子的参与。在半乳糖,ATP小分子的协助下Gal3可以捕获核内的Gal80从而解除对Gal4的抑制作用来实现GAL基因的正常诱导表达。而NADP(H)通过与Gal80的直接结合来去稳定Gal80-Gal4复合物来实现Gal4的解抑制作用,从而激活GAL基因的表达。由此看来Gal4与Gal80这两个蛋白之间的相互作用成为GAL基因的正常调控表达的关键。通过在ATP和NADP(H)合成受影响的基因缺失菌株中过量表达Gal3或是表达与Gal80相互作用减弱的Gal4的突变体看其代谢半乳糖的情况,来探讨这些基因缺失菌株的半乳糖代谢缺陷机制。实验结果显示,过量表达Gal3或是表达与Gal80相互作用减弱的Gal4的突变体均能够回补ATP合成受影响的△rip1,△atp1菌株,和NADP(H)水平降低的Δpos5和Δutr1菌株。从报告基因的水平,GAL1的转录水平以及磷酸化形式的RNA聚合酶在启动子上的占据情况以及在以葡萄糖和半乳糖为碳源的平板上的生长情况来看均能达到野生型菌的情况。说明了过量表达Gal3或是表达与Gal80相互作用减弱的Gal4的突变体均能回补ATP合成受影响的△rip1,△atp1菌株,和NADP(H)水平降低的Δpos5和Δutr1菌株。
从以上的实验结果可以推测,胞内的能量和NADP(H)水平可能改变了Gal4-Gal80间的相互作用；而这两种途径的调节作用是否具有协同效应目前还没有研究。因而本部分内容主要探讨胞内的能量水平和NADP(H)对Gal4-Gal80间的相互作用的调节。体外的实验结果显示,ATP,半乳糖和Gal3能够去稳定Gal4-Gal80复合物,而NADP(H)也能够去稳定Gal4-Gal80复合物,而NAD(H)却不能。当ATP,半乳糖和Gal3这条路径去稳定Gal4-Gal80复合物时加入一定浓度的NADP时,能够减小Gal3的用量；同样NADP(H)去稳定Gal4-Gal80复合物时加入一定浓度的ATP,半乳糖和Gal3,也可以减少NADP的用量；当这两条路径同时存在时,检测不同时间去稳定Gal4-Gal80复合物的效果表明两者具有协同性；另一方面在能量合成受影响的JN1-rip1菌株中超标达Pos5时,GAL基因的转录水平和报告基因的活性都有一定程度的恢复,可见胞内的能量和NADP(H)水平能够协同Gal80解离；对细胞能量合成受影响的Sc320-rip1菌株和胞内NADP(H)水平下降的Sc320-pos5菌株中Gal80的CHIP结果表明,在基因缺失菌株中Gal80没有发生解离。可见胞内能量和NADP(H)水平的降低改变了Gal4-Gal80之间的相互作用,从而导致GAL基因转录缺陷；在DSG1缺失菌株中Gal80的CHIP结果发现也没有有效地解离,当其转录水平在兼性厌氧条件下进一步降低时Gal80的解离进一步受到严重影响；另一方面与Gal80相互作用减弱的K23R突变体恢复了功能性mRNA的合成,其Gal80的解离作用也恢复了正常。因而Gal80从Gal4上有效解离不仅决定着GAL基因能否转录,还影响mRNA的加工过程,最终实现对整个转录过程的精细调控。
Yeast galactose metabolism genes are induced by galactose, and based on different external carbon sources there are three modes in gene expression:when on glucose, the GAL switch is repressed by various inhibition components, such as Migl,on the other hand, the transcription activator Gal4 also binds the UAS sequence with the transcription inhibitor Gal80 in its transcriptional activation (TAD); when on the glycerol/lactate or raffinose as carbon source in non-inducing conditions, the repression of the glucose-caused is relieved, but the GAL switch is remain turned off because the transcriptional activity of Gal4 is inhibited by Gal80;when on galactose as the carbon source, galactose, ATP, and the signal sensing protein Gal3 can relieve the inhibition of Gal80 in transcription activator Gal4,and at this time various transcription co-activator and RNA polymerase can be recruited on the promoter of the gal genes by the expose of Gal4-TAD. This induction is fast and efficient, within a few minutes the GAL mRNAs are up to 1000 times than in the non-inducing condition. Above all, transcription of galactose-inducible genes in yeast is regulated by the prototypical transcription activator Gal4p by interactions with Gal80,and during the GAL genes transcription, it has been also demonstrated that Gal4p becomes phosphorylated upon activation by polⅡ-associated kinases. Among the identified sites of phosphorylation within Gal4p, the most notable ones are serine 699 (S699) and serine 837 (S837),and S699 and S837 phosphorylations have been shown not to be required for Gal4p activity but as a consequence of activation in its transcriptional activity. Moreover, the stability of transcriptionally active forms of Gal4p reflected by S699 phosphorylation has been demonstrated to be regulated by a F box protein Dsgl and on non-inducing condition the phosphorylation of Gal4 in 837 serine is regulated by another F-box, Grr1. In the absence of Dsg1, ubiquitin-mediated destruction of transcription-coupled Gal4p and productive GAL mRNA synthesis was compromised, thus a Dsgl-meiated proteolysis-dependent mode of regulating Gal4p activity was proposed. On the other hand, non-proteolytic mode of regulation of Gal4p has also been reported to participate in regulating the formation of activator-promoter complexes to limit the number rounds of transcription without continued responsiveness to environmental signals.
petite mutant also referred to as a small colony, is a mutant yeast. In the sixties and seventies, researchers found some petites defect in utilization of galactose, maltose and other carbon sources, and speculated that there must be some signals that from mitochondria responsible for this phenotype. Dsgl/Mdm30 targeted for Gal4 degradation in inducing condition has been originally shown to associate with mitochondria and to be required for maintenance of fusion-competent mitochondria. Deletion of Dsgl leads to aggregated mitochondria, loss of mtDNA and a failure to respire. Besides the selective degradation of Fzo1 (GTPase) associated with the mitochondrial outer membrane fusion is also by Dsg1. In this respect, some unidentified signal(s) derived from mitochondria have been reported to be involved in the regulation of GAL gene expression. But the correlation with the galactose metabolism and mitochondrial dysfunction are not clear at the moment. About 95% of the energy needed for cell life activity is produced in mitochondria. While In the galactose metabolism, ATP directly involved in regulation of GAL gene expression that on the other hand implies the regulation role of mitochondrial function in the GAL system.; NADPH is an important coenzyme not only involved in the biological synthesis reaction is responsible for providing reducing power, and also has an important role in maintaining resistance to reactive oxygen species toxic to cell survive. And recently it has been reported that the cofactors NAD (H)/NADP (H) can directly bind the transcription inhibitor Gal80 and as a result regulate the interaction of Gal4 and Gal80:the binding of NAD (H) in Gal80 can stabilize Gal4-Gal80 relations while the binding of NADP (H) in Gal80 can destabilize the relationship between the Gal4-Gal80 facilitating GAL genes expression. As the mitochondrion is the main place to provide the reducing power NADPH, the level of intracellular NAD (H)/NADP (H) in specific regulation of GAL gene expression may have relationship with motochondria.
Saccharomyces cerevisiae is the most simple single cell model, it has readily manipulable genetic system. Since many cellular processes such as protein folding and quality control system, membrane trafficking, and cellular stress responses are highly conserved in many fundamental aspects between human and budding yeast, pathophysiological processes of quite a few neurodegenerative disorders including Creutzfeldt-Jacob, Parkinson's disease and cancer have been modeled in yeast and useful insights regarding the fundamental mechanisms have thus been gained in the past decades. At the same time, Saccharomyces cerevisiae is also one of the three major model organisms in molecular biology. This thesis work on the galactose metabolic regulation on the basis of a systematic study on the transcriptional activator Gal4, and interaction of Gal4-Gal80 was carried out to exploring the possible mechanism in the regulation of the GAL. In this paper, The major results of the thesis are as follows:
1. By analyzing different gene deletion strains and site-directed mutagenesis, or part deletion mutations of the Gal4, exploring the role of the alterations in the interaction between GAL4 and GAL80 in regulation of the yeast GAL regulon mediated by the F box protein Dsg1
To test the role of S699 phosphorylation in Dsgl-mediated GAL gene expression, transcriptional activities of Gal4 mutants bearing changes of S699 or S837 to glutamate were analyzed. Similar to wild-type Gal4p, productive activation of transcription by Gal4 S699E, S837E as well as a double mutant (S699E S837E) was severely impaired with deletion of dsgl, though a relative higher level of reporter gene expression driven by these mutants was observed as compared with wild-type Gal4 under both non-inducing and inducing conditions in the presence of Dsg1, This results suggests that S699 phosphorylation is only correlated with, but not required for, the Dsg1-mediated regulation of GAL gene activation; In contrast to previous results, accumulation of multi-ubiquitylated Gal4p was not affected by the absence of dsgl in response to galactose. Moreover, the induction defect with deletion of dsg1 was partly rescued by deletion of dnml, whose simultaneous absence has been shown to rescue the defect in mitochondria fusion caused by defective dsgl,and our results revealed that the dsg1-deleted strain displayed a slow induction phenotype and effects the early-onset expression of GAL genes in response to galactose. While the requirement for Dsg1 can be suppressed by defective GAL80 as well as by GAL4 mutations effecting Gal4p-Gal80p interaction. Also change of lysine 23 to arginine in Gal4p DBD results in a weakened interaction between Gal4p and Gal80p which may partly bypass the requirement for Dsg1 at the immediate early stage of induction.
2. By constructing and analyzing△rip1 and△atp1 gene deletion strains, the mechanism of energy state in regulation of the GAL system
In order to assess the importance of cellular energetic status on the induction of GAL regulon in response to galactose, a△rip1 strain which lacks the functional respiratory chain, and an△atp1 strain which carries a nonfunctional ATP synthase were constructed. The intracellular ATP concentration in both strains drastically decreased after the glucose-to-galactose switch. Productive activation of transcription by Gal4p as measured by the endogenous GAL1 mRNA as well as the GAL1-LacZ reporter gene expression was almost abolished in both strains in response to galactose. The lack of transcription upon induction was further found to correlate with the drastically reduced distribution of RNA polymeraseⅡ(polⅡ) across the GAL1 gene. These results indicate that maintenance of an appropriate level of intracellular energy charge is essential for the yeast cells to rapidly turn on the GAL genes through enabling the efficient Gal3p-Gal80p interaction and adapt to a new carbon substrate.
3. By constructing and analyzing△pos5 and△utr1 strains exploring the effect the mechanism of intracellular NAD (H)/NADP (H) levels in regulation GAL gene expression
To address the role of the NAD(H)/NADP(H) in regulation of the GAL system, we constructed yeasts deleted for the two major NAD kinase genes, pos5 and utr1, whose products play a critical role in determining the levels of NADP in mitochondria and cytoplasm respectively. The coding sequence of Nma2 which participates in a nuclear salvage way of NAD synthesis was also deleted as a control. The intracellular levels of both NADP and NADPH dropped dramatically in pos5 deleted strain and were slightly reduced in utr1 deleted strain. Analysis of the GAL1 transcription as well as the GAL1-LacZ reporter gene expression revealed that, in contrast to deletion of nma2, transcriptional activation in response to galactose did not occur in pos5 mutant and was severely impaired in utr1 deletion strain. Similar to ATP synthesis mutants, distribution of RNA polymeraseⅡ(polⅡ) across the GAL1 gene was drastically reduced in both mutants. To ask whether other gene targets might be affected in these mutants including△rip1 and△atp1, we examined both the transcription of and polymerase density across two other genes, ADH1 and PMA1. This analysis showed that neither the transcription nor the appearance of polⅡacross the genes was affected in these deletion mutants under both uninducing and inducing conditions. To further test the effect of changes in levels of NADP(H) on the ability of yeasts to use galactose as a carbon source, yeasts cells were spotted onto plates containing various concentrations of hydrogen peroxide. In contrast to deletion of pos5 which virtually eliminated the yeast growth on galactose, yeasts deleted for utr1 are still able to use galactose as a carbon source. Notably, the potential nonspecific, toxic effect of H2O2 is not responsible for the inability of yeast to grow on galactose media because all strains grew well on glucose media. These results suggest that an appropriate level of intracellular NADP(H) is critical for the yeast cells to effciently turn on the GAL genes.
4. Overexpression of Gal3p or Gal4p mutants with altered Gal80p-binding characteristics restores GAL gene expression in mutants deficient in ATP or NADPH synthesis
Analysis of RNA species corresponding to the 3'ends of the GAL3 gene or GAL80 gene revealed that, although the modest induction of both genes did not occur in△rip1 and△pos5 cells in response to galactose as did in wild-type, the expression levels of GAL3 and GAL80 in these yeasts were not affected under non-inducing conditions. Together with previous reports, the results indicated that the perturbed GAL switch due to the disabled dual feedback loops is not responsible for the inability of yeast to respond to galactose induction. To probe further into the possibility that the normal dissociation of Gal80p from Gal4p might be affected upon induction in mutant yeasts, we asked whether destabilizing Gal80p-Gal4p interaction rescues the defective GAL gene expression. In contrast to the induction defect displayed by pos5 or rip1 deletion in GAL80+ cells, induction of a GAL1-LacZ reporter in pos5 or rip1-deleted strains simultaneously lacking Gal80p (gal80-) was as efficient as in wild-type yeast. Moreover, activation of a GAL1-LacZ reporter gene was largely recovered after galactose induction in the mutant cells overexpressing Gal3p. Similar results were also observed for mutant cells expressing△683 or K23R, both of which have been shown to have a weakened interaction with Gal80p. This rescued activation of expression was consistent with the increased distribution of the polⅡacross the GAL1 gene. These results demonstrate that cellular ATP and NADP(H) are involved in regulating the interaction between Gal4p and Ga80p.
5. Gal3p synergizes with NADPH to dissociate Gal80p from DNA-bound Gal80p-Gal4p complex
The results above implicate that the defect in GAL gene activation in ATP or NADP(H) synthesis mutant is to a larger extent caused by the failure of Gal80p to dissociate from Gal4p. Either Gal3p or NADP(H) has been shown to destabilize the Gal80p-Gal4p interaction. To address whether NADP(H) synergizes with Gal3p in the destabilization of DNA-bound Gal4p-Gal80p complex, we performed the following experiment. A preformed DNA-Gal4p-Gal80p complex with recombinant miniGal4p and Gal80p bound to oligonucleotides containing three consensus Gal4p binding sites was incubated either with NADP alone or with Gal3p in the presence of ATP and galactose. The amount of Gal80p retained by biotin-conjugated DNA was determined by Western blot. Our results reveales that, in accordance with previous results, NADP alone caused a marked decrease in the retention of Gal80p by the DNA-bound Gal4p. In contrast, such a destabilizing effect did not occur with the addition of NAD. The preformed complex was further incubated with varying concentrations of NADP in the presence of a constant concentration of Gal3p (1 nM) plus galactose and ATP. The dissociation of Gal80p occurred at a lower concentration of NADP. Similarly, a more efficient dissociation of Gal80p from the complex was achieved in the presence of NADP with one half of the amount required for the same dissociation by Gal3p alone. On the basis of these results, we suggest that NADP(H) synergizes with ATP-Gal3p in assuring the efficient dissociation of Gal80p from Gal4p in response to galactose induction. Because the association of Gal80p with Gal4p on the promoter of GAL gene under noninducing conditions and rapid dissociation from Gal4p in response to galactose is critical for the GAL gene transcription regulation, we asked whether the association of Gal80p with Gal4p is changed in vivo with cellular ATP or NADP(H) shortage after the addition of galactose. In vivo chromatin immunoprecipitation (Chip) was used to monitor the extent of the association Gal4p and Gal80p with the UASGAL of the GAL1 promoter. In wild-type cells, the association of Gal80p with the promoter decreased dramatically upon galactose induction whereas there was almost no change for the occupancy by Gal4p before and after galactose addition. In rip1△or pos5△cells, however, the Gal80p association with the UASGAL was only slightly decreased in response to galactose.The failure of rip1△or pos5△yeasts to achieve the dissociation of Gal80p from Gal4p demonstrates that ATP and NADP(H) are physiological relevant in playing a role in regulating transcriptional activation of GAL genes in response to the change of carbon sources. Analysis of the GAL1 mRNA revealed that the efficient transcription of Gal4p target genes in the absence of dsgl was severely impaired and was correlated with increased association of Gal80p with the promoter when Adsgl cells were induced with galactose under microanaerobic conditions. These results suggest that the incomplete disscociation of Gal80p as compared with that in wild-type strain is sufficient for the transcriptional activation in the absence of dsgl. Considering that the induction defect in dsg1-null was rescued by Gal4 mutants with decreased interaction with Gal80p, we asked whether the dissociation of Gal80p as well as the distribution of Ser5-phosphorylated polⅡare restored in△dsg1 cells by Gal4p K23R. In accordance with previous results, while Ser5-phosphorylated CTD of polⅡwas dramatically reduced within the GAL1 ORF in the absence of Dsgl, robust Ser5 phosphorylation was restored by Gal4p K23R. Importantly, the dissociation of Gal80p from the promoter occurred as efficiently as in wild-type strain upon galactose addition. These results again provide evidence that the efficient dissociation of Gal80p from Gal4p not only represents a key step in determining the on-off of transcription of GAL genes in response to galactose addition, but also suggest that the appropriate spatial distribution of Gal80p relative to Gal4p may also be involved in the fine tuning of the whole transcription process including the formation of mature mRNAs.
petite mutant亦简称小菌落,是酵母菌的一种突变型。在六七十年代,就发现了小菌落在半乳糖,麦芽糖等碳源上生长的缺陷,从而提出了线粒体中有控制着对不同糖利用的因子。而诱导条件下介导Gal4降解的Dsg1还控制着与线粒体外膜融合相关的Fzo1(GTPase)的降解,Dsg1的缺失同时也造成了线粒体形态的异常。因而Dsg1对于半乳糖代谢的影响是否与线粒体的功能异常有关还不清楚；线粒体中存在着催化物质代谢和能量转换的各种酶和辅酶,因而供能物质(如糖酵解产物丙酮酸)在线粒体内能得到彻底氧化分解,生成更多的高能磷酸化合物ATP以备细胞其它生命活动需要。细胞生命活动中所需能量约有95%来自线粒体。因此,线粒体的主要功能是进行细胞的氧化供能,故有细胞内“动力工厂”之称。在半乳糖代谢过程中,ATP直接参与了GAL基因的表达调控,这也从另一方面暗示了线粒体的产能与半乳糖的正常利用有关。另一方面,NADPH是生物体的一种重要辅酶,它不仅在生物合成反应中负责提供还原力,而且对于细胞抵抗活性氧的毒害也有重要作用。最近有研究表明,辅酶因子NAD(H)/NADP(H)能够直接和转录抑制因子Gal80结合来直接调控Gal4和Gal80之间的关系：当NAD(H)与Gal80结合时,能够稳定Gal4-Gal80之间的关系；而当NADP(H)与Gal80相结合时,能够去稳定Gal4-Gal80之间的关系从而有利于GAL基因的表达。而线粒体又是提供还原力的主要场所,能为细胞抗氧化做很大的贡献。因而线粒体中的NAD(H)/NADP(H)水平是否特异性调控GAL基因的表达成为了本论文研究的一个重点,对于更深层次揭示GAL基因调控机制有着重要的作用。作为单细胞真核生物的酿酒酵母,本身不仅具有高度的遗传可操作性,而且过去几十年以其作为模式生物的研究已经使人们对一些基础的细胞生物学过程及人类一些复杂疾病,如Creutzfel-Jacob病、Parkinson's病及癌症等发生机制的认识上取得了许多关键性的进展。与此同时,酿酒酵母作为分子生物学研究的三大模式生物之一,对许多基因的转录调控相关问题的研究具有其它生物无可比拟的独特优势。本论文工作在对半乳糖代谢调控进行较为系统研究的基础上,就转录激活因子Gal4的新的活性调控方式以及Gal4-Gal80相互作用关系对GAL基因的调控上进行了系统研究,对可能的作用机制进行了初步探讨。本文主要的工作内容及结果如下：
1.通过构建不同的基因缺失菌株和Gal4的定点突变或是部分缺失突变体,从遗传学角度和体外的实验探索了Gal4-Gal80之间的相互作用模式的改变对Dsg1调控GAL基因的影响机制
通过检测Gal4的699位丝氨酸,837位丝氨酸和两个位点都突变的点突变体在野生型菌株和△dsg1菌株中的报告基因的活性显示,这两个位点的磷酸化对于Dsg1的调控没有影响,即Dsg1对于GAL基因的调控并不是通过这两个位点的磷酸化来实现的；与以前文献报道不同,在半乳糖为碳源的平板的生长实验表明△dsg1菌株与野生型菌株相比,表现出了延迟生长的表型而不是不生长,这也与△dsg1菌株中的GAL基因诱导曲线相符,很可能Dsg1只是影响了GAL基因的初期诱导。Western实验结果表明,Dsg1的缺失并没有影响Gal4的泛素化,一方面Dsgl影响GAL基因表达不是通过对Gal4的泛素化来实现的,另一方面很可能存在有其他的泛素化Gal4的泛素连接酶。由于Dsg1还特异性识别降解与线粒体外膜融合相关的蛋白Fzo1,因而Dsg1缺失造成了线粒体形态的异常。但与线粒体分裂相关的基因dnm1缺失时能够部分回补线粒体形态上的不正常,通过检测△dnm1,△dnm1△dsg1菌株中的报告基因活性发现,线粒体形态的部分恢复能够部分恢复GAL基因的诱导表达,暗示了Dsg1对GAL基因的调控可能与线粒体的功能异常有关。当在gal80缺陷菌株或是不与Gal80相互作用或相互作用减弱的Gal4的突变体中,检测报告基因的活性发现△dsgl菌株中GAL基因恢复了正常的诱导；同时体内的酵母双杂交实验和体外的实验证明了回补Dsg1缺失的Gal4的突变体确实与转录抑制因子Gal80之间的相互作用减弱了。当检测与Gal80恢复正常的相互作用的Gal4的突变体的报告基因时,Dsg1缺失所造成的GAL基因诱导缺陷又存在了,充分说明了Gal4-Gal80之间的相互作用模式改变对Dsg1调控GAL基因有很大的影响。
2.通过构建线粒体bc1复合物的RIP1基因缺失菌株和ATP合成酶的ATP1基因缺失菌株,检测当酵母细胞胞内能量状态异常时对于GAL基因表达的影响及其机制
通过检测△rip1,△atp1菌株中的报告基因活性,结果显示GAL基因没有表达。提取△rip1,△atp1菌株中的mRNA,检测GAL1的(?)nRNA是否有转录,结果表明,△rip1,△atp1菌株中没有mRNA生成；CHIP实验进一步表明无论是RNA聚合酶还是羧基末端功能区5-丝氨酸磷酸化的RNA聚合酶都不能正常的结合启动子,造成GAL基因不能转录,从而△rip1菌株不能够正常的利用半乳糖。为了确定△rip1和△atp1基因是否真正影响了细胞内的ATP水平,我检测了基因缺失菌株中ATP浓度,结果显示胞内的ATP浓度与野生型菌株相比确实下降了很多；由此可以推断△rip1和△atp1由于影响了ATP的合成,造成了胞内ATP浓度的降低从而引起了GAL基因的转录缺陷。因为胞内各种代谢途径都会受到ATP水平的影响,因而RIP1和ATP1基因的缺失对半乳糖代谢的影响是否具有特异性?通过进一步对组成型表达基因ADH1和PMA1的mRNA的检测,结果显示ADH1和PAA1在野生型和△rip1和△atp1菌株均能正常的转录；葡萄糖和半乳糖为碳源的平板生长结果表明,野生型和△rip1和△atp1菌株均能正常的在以葡萄糖为碳源的平板生长,而以半乳糖为碳源的平板上△rip1和△atp1菌株均不能正常的生长。以上结果表明了RIP1和ATP1基因的缺失引起的胞内ATP水平的降低能够特异性的导致GAL基因转录缺陷。
3.通过构建线粒体中NAD(H)激酶POS5基因缺失菌株和细胞质中NAD激酶UTRl的基因缺失菌株,检测酵母细胞胞内NAD(H)／NADP(H)水平对GAL基因表达的影响及其机制
通过检测Δpos5和Δutr1菌株中的报告基因活性,结果显示GAL基因没有表达。提取Δpos5和Δutr1菌株中的mRNA,检测GAL1的mRNA是否有转录,结果表明,Δpos5和Δutr1菌株中没有mRNA生成；CHIP实验进一步表明无论是RNA聚合酶还是羧基末端功能区5-丝氨酸磷酸化的RNA聚合酶都不能正常的结合启动子,造成GAL基因不能转录,从而Δpos5菌株不能够正常的利用半乳糖。为了确定POS5和UTR1基因是否真正影响了细胞内NAD(H)／NADP(H)的水平,通过检测基因缺失菌株中NAD(H)／NADP(H)浓度,结果显示胞内的NAD(H)浓度与野生型菌株相比基本没有影响,而胞内NADP(H)的水平确实下降了很多,分别以葡萄糖和半乳糖为碳源的平板上对不同浓度H202的敏感性实验说明了Δpos5和Δutr1菌株与野生型相比抗氧化能力有了很大的降低,这很可能与胞内NADP(H)的水平下降有关；由此可以推断Δpos5和Δutr1基因缺失菌株由于影响了胞内NADP(H)浓度,造成了GAL基因的转录缺陷。因为胞内各种代谢途径都会受到NADP(H)水平的影响,因而POS5和UTR1基因的缺失对半乳糖代谢的影响是否具有特异性?通过进一步对组成型表达基因ADH1和PMA1的mRNA的检测,结果显示ADH1和PMA1在野生型和Δpos5和Δutr1菌株中均能正常的转录；葡萄糖和半乳糖为碳源的平板生长结果表明,野生型,Δpos5和Δutr1菌株均能正常的在以葡萄糖为碳源的平板生长,而以半乳糖为碳源的平板上Δpos5和Δutr1菌株均不能正常的生长。以上结果表明了POS5和UTR1基因的缺失引起的胞内NADP(H)水平的降低能够特异性的导致GAL基因转录缺陷。
4.在ATP合成途径基因和NAD(H)激酶基因缺失菌中过量表达Gal3或与Gal80相互作用改变的Gal4突变体与半乳糖代谢功能的恢复机制的研究
在半乳糖正常的代谢过程中,GAL基因的正常调控表达是必须的。GAL基因的诱导表达受转录激活因子Gal4和转录抑制因子Gal80,信号感应蛋白Gal3还有半乳糖,ATP小分子的参与。在半乳糖,ATP小分子的协助下Gal3可以捕获核内的Gal80从而解除对Gal4的抑制作用来实现GAL基因的正常诱导表达。而NADP(H)通过与Gal80的直接结合来去稳定Gal80-Gal4复合物来实现Gal4的解抑制作用,从而激活GAL基因的表达。由此看来Gal4与Gal80这两个蛋白之间的相互作用成为GAL基因的正常调控表达的关键。通过在ATP和NADP(H)合成受影响的基因缺失菌株中过量表达Gal3或是表达与Gal80相互作用减弱的Gal4的突变体看其代谢半乳糖的情况,来探讨这些基因缺失菌株的半乳糖代谢缺陷机制。实验结果显示,过量表达Gal3或是表达与Gal80相互作用减弱的Gal4的突变体均能够回补ATP合成受影响的△rip1,△atp1菌株,和NADP(H)水平降低的Δpos5和Δutr1菌株。从报告基因的水平,GAL1的转录水平以及磷酸化形式的RNA聚合酶在启动子上的占据情况以及在以葡萄糖和半乳糖为碳源的平板上的生长情况来看均能达到野生型菌的情况。说明了过量表达Gal3或是表达与Gal80相互作用减弱的Gal4的突变体均能回补ATP合成受影响的△rip1,△atp1菌株,和NADP(H)水平降低的Δpos5和Δutr1菌株。
从以上的实验结果可以推测,胞内的能量和NADP(H)水平可能改变了Gal4-Gal80间的相互作用；而这两种途径的调节作用是否具有协同效应目前还没有研究。因而本部分内容主要探讨胞内的能量水平和NADP(H)对Gal4-Gal80间的相互作用的调节。体外的实验结果显示,ATP,半乳糖和Gal3能够去稳定Gal4-Gal80复合物,而NADP(H)也能够去稳定Gal4-Gal80复合物,而NAD(H)却不能。当ATP,半乳糖和Gal3这条路径去稳定Gal4-Gal80复合物时加入一定浓度的NADP时,能够减小Gal3的用量；同样NADP(H)去稳定Gal4-Gal80复合物时加入一定浓度的ATP,半乳糖和Gal3,也可以减少NADP的用量；当这两条路径同时存在时,检测不同时间去稳定Gal4-Gal80复合物的效果表明两者具有协同性；另一方面在能量合成受影响的JN1-rip1菌株中超标达Pos5时,GAL基因的转录水平和报告基因的活性都有一定程度的恢复,可见胞内的能量和NADP(H)水平能够协同Gal80解离；对细胞能量合成受影响的Sc320-rip1菌株和胞内NADP(H)水平下降的Sc320-pos5菌株中Gal80的CHIP结果表明,在基因缺失菌株中Gal80没有发生解离。可见胞内能量和NADP(H)水平的降低改变了Gal4-Gal80之间的相互作用,从而导致GAL基因转录缺陷；在DSG1缺失菌株中Gal80的CHIP结果发现也没有有效地解离,当其转录水平在兼性厌氧条件下进一步降低时Gal80的解离进一步受到严重影响；另一方面与Gal80相互作用减弱的K23R突变体恢复了功能性mRNA的合成,其Gal80的解离作用也恢复了正常。因而Gal80从Gal4上有效解离不仅决定着GAL基因能否转录,还影响mRNA的加工过程,最终实现对整个转录过程的精细调控。
Yeast galactose metabolism genes are induced by galactose, and based on different external carbon sources there are three modes in gene expression:when on glucose, the GAL switch is repressed by various inhibition components, such as Migl,on the other hand, the transcription activator Gal4 also binds the UAS sequence with the transcription inhibitor Gal80 in its transcriptional activation (TAD); when on the glycerol/lactate or raffinose as carbon source in non-inducing conditions, the repression of the glucose-caused is relieved, but the GAL switch is remain turned off because the transcriptional activity of Gal4 is inhibited by Gal80;when on galactose as the carbon source, galactose, ATP, and the signal sensing protein Gal3 can relieve the inhibition of Gal80 in transcription activator Gal4,and at this time various transcription co-activator and RNA polymerase can be recruited on the promoter of the gal genes by the expose of Gal4-TAD. This induction is fast and efficient, within a few minutes the GAL mRNAs are up to 1000 times than in the non-inducing condition. Above all, transcription of galactose-inducible genes in yeast is regulated by the prototypical transcription activator Gal4p by interactions with Gal80,and during the GAL genes transcription, it has been also demonstrated that Gal4p becomes phosphorylated upon activation by polⅡ-associated kinases. Among the identified sites of phosphorylation within Gal4p, the most notable ones are serine 699 (S699) and serine 837 (S837),and S699 and S837 phosphorylations have been shown not to be required for Gal4p activity but as a consequence of activation in its transcriptional activity. Moreover, the stability of transcriptionally active forms of Gal4p reflected by S699 phosphorylation has been demonstrated to be regulated by a F box protein Dsgl and on non-inducing condition the phosphorylation of Gal4 in 837 serine is regulated by another F-box, Grr1. In the absence of Dsg1, ubiquitin-mediated destruction of transcription-coupled Gal4p and productive GAL mRNA synthesis was compromised, thus a Dsgl-meiated proteolysis-dependent mode of regulating Gal4p activity was proposed. On the other hand, non-proteolytic mode of regulation of Gal4p has also been reported to participate in regulating the formation of activator-promoter complexes to limit the number rounds of transcription without continued responsiveness to environmental signals.
petite mutant also referred to as a small colony, is a mutant yeast. In the sixties and seventies, researchers found some petites defect in utilization of galactose, maltose and other carbon sources, and speculated that there must be some signals that from mitochondria responsible for this phenotype. Dsgl/Mdm30 targeted for Gal4 degradation in inducing condition has been originally shown to associate with mitochondria and to be required for maintenance of fusion-competent mitochondria. Deletion of Dsgl leads to aggregated mitochondria, loss of mtDNA and a failure to respire. Besides the selective degradation of Fzo1 (GTPase) associated with the mitochondrial outer membrane fusion is also by Dsg1. In this respect, some unidentified signal(s) derived from mitochondria have been reported to be involved in the regulation of GAL gene expression. But the correlation with the galactose metabolism and mitochondrial dysfunction are not clear at the moment. About 95% of the energy needed for cell life activity is produced in mitochondria. While In the galactose metabolism, ATP directly involved in regulation of GAL gene expression that on the other hand implies the regulation role of mitochondrial function in the GAL system.; NADPH is an important coenzyme not only involved in the biological synthesis reaction is responsible for providing reducing power, and also has an important role in maintaining resistance to reactive oxygen species toxic to cell survive. And recently it has been reported that the cofactors NAD (H)/NADP (H) can directly bind the transcription inhibitor Gal80 and as a result regulate the interaction of Gal4 and Gal80:the binding of NAD (H) in Gal80 can stabilize Gal4-Gal80 relations while the binding of NADP (H) in Gal80 can destabilize the relationship between the Gal4-Gal80 facilitating GAL genes expression. As the mitochondrion is the main place to provide the reducing power NADPH, the level of intracellular NAD (H)/NADP (H) in specific regulation of GAL gene expression may have relationship with motochondria.
Saccharomyces cerevisiae is the most simple single cell model, it has readily manipulable genetic system. Since many cellular processes such as protein folding and quality control system, membrane trafficking, and cellular stress responses are highly conserved in many fundamental aspects between human and budding yeast, pathophysiological processes of quite a few neurodegenerative disorders including Creutzfeldt-Jacob, Parkinson's disease and cancer have been modeled in yeast and useful insights regarding the fundamental mechanisms have thus been gained in the past decades. At the same time, Saccharomyces cerevisiae is also one of the three major model organisms in molecular biology. This thesis work on the galactose metabolic regulation on the basis of a systematic study on the transcriptional activator Gal4, and interaction of Gal4-Gal80 was carried out to exploring the possible mechanism in the regulation of the GAL. In this paper, The major results of the thesis are as follows:
1. By analyzing different gene deletion strains and site-directed mutagenesis, or part deletion mutations of the Gal4, exploring the role of the alterations in the interaction between GAL4 and GAL80 in regulation of the yeast GAL regulon mediated by the F box protein Dsg1
To test the role of S699 phosphorylation in Dsgl-mediated GAL gene expression, transcriptional activities of Gal4 mutants bearing changes of S699 or S837 to glutamate were analyzed. Similar to wild-type Gal4p, productive activation of transcription by Gal4 S699E, S837E as well as a double mutant (S699E S837E) was severely impaired with deletion of dsgl, though a relative higher level of reporter gene expression driven by these mutants was observed as compared with wild-type Gal4 under both non-inducing and inducing conditions in the presence of Dsg1, This results suggests that S699 phosphorylation is only correlated with, but not required for, the Dsg1-mediated regulation of GAL gene activation; In contrast to previous results, accumulation of multi-ubiquitylated Gal4p was not affected by the absence of dsgl in response to galactose. Moreover, the induction defect with deletion of dsg1 was partly rescued by deletion of dnml, whose simultaneous absence has been shown to rescue the defect in mitochondria fusion caused by defective dsgl,and our results revealed that the dsg1-deleted strain displayed a slow induction phenotype and effects the early-onset expression of GAL genes in response to galactose. While the requirement for Dsg1 can be suppressed by defective GAL80 as well as by GAL4 mutations effecting Gal4p-Gal80p interaction. Also change of lysine 23 to arginine in Gal4p DBD results in a weakened interaction between Gal4p and Gal80p which may partly bypass the requirement for Dsg1 at the immediate early stage of induction.
2. By constructing and analyzing△rip1 and△atp1 gene deletion strains, the mechanism of energy state in regulation of the GAL system
In order to assess the importance of cellular energetic status on the induction of GAL regulon in response to galactose, a△rip1 strain which lacks the functional respiratory chain, and an△atp1 strain which carries a nonfunctional ATP synthase were constructed. The intracellular ATP concentration in both strains drastically decreased after the glucose-to-galactose switch. Productive activation of transcription by Gal4p as measured by the endogenous GAL1 mRNA as well as the GAL1-LacZ reporter gene expression was almost abolished in both strains in response to galactose. The lack of transcription upon induction was further found to correlate with the drastically reduced distribution of RNA polymeraseⅡ(polⅡ) across the GAL1 gene. These results indicate that maintenance of an appropriate level of intracellular energy charge is essential for the yeast cells to rapidly turn on the GAL genes through enabling the efficient Gal3p-Gal80p interaction and adapt to a new carbon substrate.
3. By constructing and analyzing△pos5 and△utr1 strains exploring the effect the mechanism of intracellular NAD (H)/NADP (H) levels in regulation GAL gene expression
To address the role of the NAD(H)/NADP(H) in regulation of the GAL system, we constructed yeasts deleted for the two major NAD kinase genes, pos5 and utr1, whose products play a critical role in determining the levels of NADP in mitochondria and cytoplasm respectively. The coding sequence of Nma2 which participates in a nuclear salvage way of NAD synthesis was also deleted as a control. The intracellular levels of both NADP and NADPH dropped dramatically in pos5 deleted strain and were slightly reduced in utr1 deleted strain. Analysis of the GAL1 transcription as well as the GAL1-LacZ reporter gene expression revealed that, in contrast to deletion of nma2, transcriptional activation in response to galactose did not occur in pos5 mutant and was severely impaired in utr1 deletion strain. Similar to ATP synthesis mutants, distribution of RNA polymeraseⅡ(polⅡ) across the GAL1 gene was drastically reduced in both mutants. To ask whether other gene targets might be affected in these mutants including△rip1 and△atp1, we examined both the transcription of and polymerase density across two other genes, ADH1 and PMA1. This analysis showed that neither the transcription nor the appearance of polⅡacross the genes was affected in these deletion mutants under both uninducing and inducing conditions. To further test the effect of changes in levels of NADP(H) on the ability of yeasts to use galactose as a carbon source, yeasts cells were spotted onto plates containing various concentrations of hydrogen peroxide. In contrast to deletion of pos5 which virtually eliminated the yeast growth on galactose, yeasts deleted for utr1 are still able to use galactose as a carbon source. Notably, the potential nonspecific, toxic effect of H2O2 is not responsible for the inability of yeast to grow on galactose media because all strains grew well on glucose media. These results suggest that an appropriate level of intracellular NADP(H) is critical for the yeast cells to effciently turn on the GAL genes.
4. Overexpression of Gal3p or Gal4p mutants with altered Gal80p-binding characteristics restores GAL gene expression in mutants deficient in ATP or NADPH synthesis
Analysis of RNA species corresponding to the 3'ends of the GAL3 gene or GAL80 gene revealed that, although the modest induction of both genes did not occur in△rip1 and△pos5 cells in response to galactose as did in wild-type, the expression levels of GAL3 and GAL80 in these yeasts were not affected under non-inducing conditions. Together with previous reports, the results indicated that the perturbed GAL switch due to the disabled dual feedback loops is not responsible for the inability of yeast to respond to galactose induction. To probe further into the possibility that the normal dissociation of Gal80p from Gal4p might be affected upon induction in mutant yeasts, we asked whether destabilizing Gal80p-Gal4p interaction rescues the defective GAL gene expression. In contrast to the induction defect displayed by pos5 or rip1 deletion in GAL80+ cells, induction of a GAL1-LacZ reporter in pos5 or rip1-deleted strains simultaneously lacking Gal80p (gal80-) was as efficient as in wild-type yeast. Moreover, activation of a GAL1-LacZ reporter gene was largely recovered after galactose induction in the mutant cells overexpressing Gal3p. Similar results were also observed for mutant cells expressing△683 or K23R, both of which have been shown to have a weakened interaction with Gal80p. This rescued activation of expression was consistent with the increased distribution of the polⅡacross the GAL1 gene. These results demonstrate that cellular ATP and NADP(H) are involved in regulating the interaction between Gal4p and Ga80p.
5. Gal3p synergizes with NADPH to dissociate Gal80p from DNA-bound Gal80p-Gal4p complex
The results above implicate that the defect in GAL gene activation in ATP or NADP(H) synthesis mutant is to a larger extent caused by the failure of Gal80p to dissociate from Gal4p. Either Gal3p or NADP(H) has been shown to destabilize the Gal80p-Gal4p interaction. To address whether NADP(H) synergizes with Gal3p in the destabilization of DNA-bound Gal4p-Gal80p complex, we performed the following experiment. A preformed DNA-Gal4p-Gal80p complex with recombinant miniGal4p and Gal80p bound to oligonucleotides containing three consensus Gal4p binding sites was incubated either with NADP alone or with Gal3p in the presence of ATP and galactose. The amount of Gal80p retained by biotin-conjugated DNA was determined by Western blot. Our results reveales that, in accordance with previous results, NADP alone caused a marked decrease in the retention of Gal80p by the DNA-bound Gal4p. In contrast, such a destabilizing effect did not occur with the addition of NAD. The preformed complex was further incubated with varying concentrations of NADP in the presence of a constant concentration of Gal3p (1 nM) plus galactose and ATP. The dissociation of Gal80p occurred at a lower concentration of NADP. Similarly, a more efficient dissociation of Gal80p from the complex was achieved in the presence of NADP with one half of the amount required for the same dissociation by Gal3p alone. On the basis of these results, we suggest that NADP(H) synergizes with ATP-Gal3p in assuring the efficient dissociation of Gal80p from Gal4p in response to galactose induction. Because the association of Gal80p with Gal4p on the promoter of GAL gene under noninducing conditions and rapid dissociation from Gal4p in response to galactose is critical for the GAL gene transcription regulation, we asked whether the association of Gal80p with Gal4p is changed in vivo with cellular ATP or NADP(H) shortage after the addition of galactose. In vivo chromatin immunoprecipitation (Chip) was used to monitor the extent of the association Gal4p and Gal80p with the UASGAL of the GAL1 promoter. In wild-type cells, the association of Gal80p with the promoter decreased dramatically upon galactose induction whereas there was almost no change for the occupancy by Gal4p before and after galactose addition. In rip1△or pos5△cells, however, the Gal80p association with the UASGAL was only slightly decreased in response to galactose.The failure of rip1△or pos5△yeasts to achieve the dissociation of Gal80p from Gal4p demonstrates that ATP and NADP(H) are physiological relevant in playing a role in regulating transcriptional activation of GAL genes in response to the change of carbon sources. Analysis of the GAL1 mRNA revealed that the efficient transcription of Gal4p target genes in the absence of dsgl was severely impaired and was correlated with increased association of Gal80p with the promoter when Adsgl cells were induced with galactose under microanaerobic conditions. These results suggest that the incomplete disscociation of Gal80p as compared with that in wild-type strain is sufficient for the transcriptional activation in the absence of dsgl. Considering that the induction defect in dsg1-null was rescued by Gal4 mutants with decreased interaction with Gal80p, we asked whether the dissociation of Gal80p as well as the distribution of Ser5-phosphorylated polⅡare restored in△dsg1 cells by Gal4p K23R. In accordance with previous results, while Ser5-phosphorylated CTD of polⅡwas dramatically reduced within the GAL1 ORF in the absence of Dsgl, robust Ser5 phosphorylation was restored by Gal4p K23R. Importantly, the dissociation of Gal80p from the promoter occurred as efficiently as in wild-type strain upon galactose addition. These results again provide evidence that the efficient dissociation of Gal80p from Gal4p not only represents a key step in determining the on-off of transcription of GAL genes in response to galactose addition, but also suggest that the appropriate spatial distribution of Gal80p relative to Gal4p may also be involved in the fine tuning of the whole transcription process including the formation of mature mRNAs.
引文
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