Chien Y, Helmann J D, Zinder S H
Section of Microbiology, Cornell University, Ithaca, New York 14853, USA.
J Bacteriol. 1998 May;180(10):2723-8. doi: 10.1128/JB.180.10.2723-2728.1998.
Transcription initiation in Archaea (archaebacteria) resembles the eucaryotic process, having been shown to involve TATA box-like promoter regions as well as TATA-binding protein and TFIIB homologs. However, little is known about transcription regulation in archaea. We have previously demonstrated that transcripts of nifHDK2 genes, encoding Methanosarcina barkeri nitrogenase, are present in N2-grown cells but not in ammonium-grown cells, indicating that nif transcription is regulated by the nitrogen source. In this study, we detected proteins in M. barkeri cell extracts that bind specifically to DNA containing the putative promoter region of nifHDK2. No binding was found when the promoter region was deleted from the DNA. A competition assay showed that the methyl coenzyme M reductase (mcr) promoter region DNA and the nifH2 promoter region DNA competed for a common factor(s). There was no binding to the nifH2 promoter region by extracts of ammonium-grown cells, but there was binding by these extracts to promoter regions for mcr genes, which are presumably constitutively expressed. Interestingly, extracts of ammonium-grown cells inhibited binding to the nif promoter region by extracts of N2-grown cells. Fractionation of extracts of ammonium-grown cells with a heparin-Sepharose column resolved them into a fraction eluting at 0 M NaCl, which inhibited binding by extracts of N2-grown cells, and a fraction eluting at 0.5 to 0.75 M NaCl, which showed binding to the promoter region. These results are congruent with a model for regulation of nif gene expression in M. barkeri in which a substance present in ammonium-grown cells inhibits DNA binding by a transcription-associated protein or proteins.
古菌（古细菌）中的转录起始类似于真核生物的过程，已显示其涉及类似TATA框的启动子区域以及TATA结合蛋白和TFIIB同源物。然而，关于古菌中的转录调控知之甚少。我们之前已经证明，编码巴氏甲烷八叠球菌固氮酶的nifHDK2基因的转录本存在于以N₂生长的细胞中，而不存在于以铵生长的细胞中，这表明nif转录受氮源调控。在本研究中，我们在巴氏甲烷八叠球菌细胞提取物中检测到了能与含有nifHDK2假定启动子区域的DNA特异性结合的蛋白质。当从DNA中删除启动子区域时，未发现结合。竞争试验表明，甲基辅酶M还原酶（mcr）启动子区域DNA和nifH2启动子区域DNA竞争一种共同因子。以铵生长的细胞提取物不与nifH2启动子区域结合，但这些提取物与mcr基因的启动子区域结合，mcr基因推测是组成型表达的。有趣的是，以铵生长的细胞提取物抑制了以N₂生长的细胞提取物与nif启动子区域的结合。用肝素 - 琼脂糖柱对以铵生长的细胞提取物进行分级分离，将其分离为在0 M NaCl时洗脱的级分，该级分抑制以N₂生长的细胞提取物的结合，以及在0.5至0.75 M NaCl时洗脱的级分，该级分显示与启动子区域结合。这些结果与巴氏甲烷八叠球菌中nif基因表达调控的模型一致，即在以铵生长的细胞中存在的一种物质抑制一种或多种转录相关蛋白与DNA的结合。
