Wenzel T J, Zuurmond A M, Bergmans A, van den Berg J A, Steensma H Y
Department of Molecular and Cellular Biology, Leiden University, The Netherlands.
Yeast. 1994 Mar;10(3):297-308. doi: 10.1002/yea.320100303.
The location and sequence of the PDA1 gene, encoding the E1 alpha subunit of the pyruvate dehydrogenase (PDH) complex from Saccharomyces cerevisiae, were determined. The PDA1 gene was located on a 6.2 kb fragment of chromosome V, approximately 18 kb centromere distal to RAD3. Consistent with this, the PDA1 gene was genetically mapped at 4 cM from RAD3. A part of the 6.2 kb fragment of chromosome V was sequenced. The nucleotide sequence contained the PDA1 open reading frame and the entire putative promoter. Computer analysis revealed a putative GCN4 binding motif in the PDA1 promoter. The presence of transcriptional elements was experimentally determined by deletion analysis. To this end, ExoIII deletions were constructed in the 5' to 3' direction of the PDA1 promoter and effects on transcription were determined by Northern analysis. Transcription was unaffected upon deletion to position -190 relative to the ATG start codon. Deletions from position -148 and beyond, however, reduced promoter activity at least 40-fold. Apparently the 42 bp between nucleotides -190 and -148 contain an element essential for transcription. Inactivation of the PDA1 promoter could not be attributed to deletions of a recognizable TATA element or any known yeast regulatory motifs. The possible role of the CCCTT sequence present in the 42 bp region and also in the promoters of the other genes encoding subunits of the PDH complex is discussed.
已确定了酿酒酵母丙酮酸脱氢酶（PDH）复合体E1α亚基的编码基因PDA1的位置和序列。PDA1基因位于第五条染色体的一个6.2kb片段上，距离RAD3着丝粒约18kb远。与此一致的是，PDA1基因在遗传图谱上位于距RAD3 4厘摩处。对第五条染色体6.2kb片段的一部分进行了测序。核苷酸序列包含PDA1开放阅读框和整个推测的启动子。计算机分析显示PDA1启动子中有一个推测的GCN4结合基序。转录元件的存在通过缺失分析进行了实验确定。为此，在PDA1启动子的5'至3'方向构建了ExoIII缺失，并通过Northern分析确定其对转录的影响。相对于ATG起始密码子缺失到-190位置时转录不受影响。然而，从-148位置及以后的缺失使启动子活性降低了至少40倍。显然，-190和-148核苷酸之间的42bp包含一个转录必需的元件。PDA1启动子的失活不能归因于可识别的TATA元件或任何已知酵母调控基序的缺失。讨论了42bp区域中存在的CCCTT序列以及其他编码PDH复合体亚基的基因启动子中该序列的可能作用。
