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首页> 外文期刊>Molecular and Cellular Biology >Structure and regulation of KGD2, the structural gene for yeast dihydrolipoyl transsuccinylase.
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Structure and regulation of KGD2, the structural gene for yeast dihydrolipoyl transsuccinylase.

机译：KGD2的结构和调控，KGD2是酵母二氢脂酰转琥珀酰酶的结构基因。

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Yeast mutants assigned to the pet complementation group G104 were found to lack alpha-ketoglutarate dehydrogenase activity as a result of mutations in the dihydrolipoyl transsuccinylase (KE2) component of the complex. The nuclear gene KGD2, coding for yeast KE2, was cloned by transformation of E250/U6, a G104 mutant, with a yeast genomic library. Analysis of the KGD2 sequence revealed an open reading frame encoding a protein with a molecular weight of 52,375 and 42% identities to the KE2 component of Escherichia coli alpha-ketoglutarate dehydrogenase complex. Disruption of the chromosomal copy of KGD2 in a respiratory-competent haploid yeast strain elicited a growth phenotype similar to that of G104 mutants and abolished the ability to mitochondria to catalyze the reduction of NAD+ by alpha-ketoglutarate. The expression of KGD2 was transcriptionally regulated by glucose. Northern (RNA) analysis of poly(A)+ RNA indicated the existence of two KGD2 transcripts differing in length by 150 nucleotides. The concentrations of both RNAs were at least 10 times lower in glucose (repressed)- than in galactose (derepressed)-grown cells. Different 5'-flanking regions of KGD2 were fused to the lacZ gene of E. coli in episomal plasmids, and the resultant constructs were tested for expression of beta-galactosidase in wild-type yeast cells and in hap2 and hap3 mutants. Results of the lacZ fusion assays indicated that transcription of KGD2 is activated by the HAP2 and HAP3 proteins. The regulated expression of KGD2 was found to depend on sequences that map to a region 244 to 484 nucleotides upstream of the structural gene. This region contains two short sequence elements that differ by one nucleotide from the consensus core (5'-TN[A/G]TTGGT-3') that has been proposed to be essential for binding of the HAP activation complex. These data together with earlier reports on the regulation of the KGD1 and LPD1 genes for the alpha-ketoglutarate and dihydrolipoyl dehydrogenases indicate that all three enzyme components of the complex are catabolite repressed and subject to positive regulation by the HAP2 and HAP3 proteins.
机译：由于该复合物的二氢脂酰转琥珀酰化酶（KE2）组件发生突变，发现分配给宠物互补组G104的酵母突变体缺乏α-酮戊二酸脱氢酶活性。通过用酵母基因组文库转化E250 / U6（一种G104突变体），克隆了编码酵母KE2的核基因KGD2。对KGD2序列的分析揭示了一个开放阅读框，该阅读框编码一种分子量为52,375的蛋白质​​，与大肠杆菌α-酮戊二酸脱氢酶复合物的KE2成分具有42％的同一性。具有呼吸功能的单倍体酵母菌株中KGD2染色体拷贝的破坏引起类似于G104突变体的生长表型，并消除了线粒体催化α-酮戊二酸催化NAD +还原的能力。 KGD2的表达受葡萄糖转录调控。对poly（A）+ RNA的Northern（RNA）分析表明存在两个长度相差150个核苷酸的KGD2转录本。葡萄糖（抑制）生长的两种RNA的浓度至少比半乳糖（抑制）生长的细胞低10倍。在游离质粒中将KGD2的不同5'侧翼区与大肠杆菌的lacZ基因融合，并测试所得构建体在野生型酵母细胞以及hap2和hap3突变体中β-半乳糖苷酶的表达。 lacZ融合测定的结果表明，KGD2的转录被HAP2和HAP3蛋白激活。发现KGD2的调节表达取决于序列，该序列映射到结构基因上游的244至484个核苷酸区域。该区域包含两个短序列元件，该两个短序列元件与共有核心（5'-TN [A / G] TTGGT-3'）相差一个核苷酸，而共有核心被认为是结合HAP激活复合物必不可少的。这些数据以及有关α-酮戊二酸和二氢脂酰脱氢酶的KGD1和LPD1基因调控的早期报道表明，复合物的所有三种酶成分均被分解代谢物抑制，并受到HAP2和HAP3蛋白的正调控。

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