Sowers K R, Thai T T, Gunsalus R P
Department of Microbiology and Molecular Genetics, University of California, Los Angeles 90024.
J Biol Chem. 1993 Nov 5;268(31):23172-8.
The mechanisms of gene regulation in the phylogenetic domain Archaea are not yet understood. To examine the expression of a gene encoding a highly regulated catabolic enzyme from the methanogenic archaea, a Methanosarcina thermophila lambda gt11 chromosomal library was probed with antiserum prepared against the 89-kDa subunit of carbon monoxide dehydrogenase, an enzyme which is required for growth and methanogenesis from acetate. A 2.3-kilobase DNA fragment was isolated that encoded 300 bases of the 5'-end of cdhA, the gene which encodes the 89-kDa subunit, and 2 kilobases upstream of cdhA that included an upstream open reading frame (ORF1). Primer extension analyses determined that cdhA and ORF1 each had a single transcriptional initiation site located 370 and 9 nucleotides, respectively, 5' of the putative translation initiation codons for cdhA and ORF1. Each promoter element had sequence similarity to a consensus archaeal promoter sequence. Three discrete mRNA cdhA transcripts of 9.5, 5.6, and 4.8 kilobases and one mRNA ORF1 transcript of < 2 kilobases were identified. All four transcripts were optimally expressed in cells grown with acetate, while growth with the more energetically favorable substrates methanol and trimethylamine caused a significant reduction in levels of the cdhA and ORF1 mRNA's. The half-lives of the 5' ends of the three cdhA transcripts and entire ORF1 mRNA transcript were approximately 2 min upon addition of methanol to cells growing exponentially in medium that contained acetate. Results of this study demonstrate that transcription of both cdhA and ORF1 is highly regulated in response to substrate by this methanogenic archaeon.
古生菌系统发育域中的基因调控机制尚未明确。为了研究产甲烷古生菌中一种编码高度调控的分解代谢酶的基因表达情况，用针对一氧化碳脱氢酶89 kDa亚基制备的抗血清对嗜热甲烷八叠球菌λ gt11染色体文库进行了筛选，一氧化碳脱氢酶是乙酸盐生长和产甲烷过程中所需的一种酶。分离出一个2.3千碱基的DNA片段，其编码了cdhA（编码89 kDa亚基的基因）5'端的300个碱基以及cdhA上游2千碱基，其中包括一个上游开放阅读框（ORF1）。引物延伸分析确定，cdhA和ORF1各自有一个单一的转录起始位点，分别位于cdhA和ORF1推定翻译起始密码子5'端的370和9个核苷酸处。每个启动子元件与古生菌启动子共有序列具有序列相似性。鉴定出了三种分别为9.5、5.6和4.8千碱基的离散cdhA mRNA转录本以及一种小于2千碱基的ORF1 mRNA转录本。所有四种转录本在以乙酸盐生长的细胞中表达最佳，而以能量更有利的底物甲醇和三甲胺生长则导致cdhA和ORF1 mRNA水平显著降低。在含有乙酸盐的培养基中指数生长的细胞中加入甲醇后，三种cdhA转录本5'端以及整个ORF1 mRNA转录本的半衰期约为2分钟。本研究结果表明，这种产甲烷古生菌中cdhA和ORF1的转录均受到底物的高度调控。
